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Applied Biosystems StepOne™ and StepOnePlus™ Real

1. RNA Quantitation Using 1 Step Reagent Kit Part Number RT PCR TagMan reagents TaqMan One Step RT PCR Master Mix 4309169 Reagents Kit TagMan EZ RT PCR Core Reagents N808 0236 Note Use the TagMan EZ RT PCR Core Reagents when a high temperature RT step is required SYBR Green Power SYBR Green RT PCR Reagents Kit 4368711 reagents SYBR Green RT PCR Reagents 4310179 RNA Quantitation Using 2 Step Reagent Step Kit Part Number RT PCR TaqMan PCR step only TaqMan Gene Expression Master 4369016 reagents Mix 1 Pack 1 x 5 mL 200 reactions TaqMan Gene Expression Master 4369542 Mix 10 Pack 10 x 5 mL 2000 reactions TaqMan 2X Universal PCR Master 4304437 Mix TagMan Fast Universal PCR Master 4352042 Mix 2X No AmpErase UNG RT step only High Capacity cDNA Reverse 4374966 Transcription Kit TagMan Reverse Transcription N808 234 Reagents Both RT and TaqMan Gold RT PCR kit N808 232 PCR steps SYBR Green PCR step only Power SYBR Green PCR Master 4367659 reagents Mix SYBR Green PCR Master Mix 4309155 RT step only High Capacity cDNA Reverse 4374966 Transcription Kit TaqMan Reverse Transcription N808 234 Reagents Both RT and Power SYBR Green RT PCR 4368711 PCR steps Reagents Kit SYBR Green RT PCR Reagents 4310179 D 4 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Genotyping Experiments Pre Designed Validated A
2. DNA or cDNA Quantitation Reagent Kit Part Number TagMan reagents TaqMan Gene Expression Master Mix 1 Pack 4369016 1 x 5 mL 200 reactions TagMan Gene Expression Master Mix 10 Pack 4369542 10 x 5 mL 2000 reactions TagMan Fast Universal PCR Master Mix 2X No 4352042 AmpErase UNG 250 Reactions TaqMan 2X Universal PCR Master Mix 200 4304437 Reactions TaqMan 2X Universal PCR Master Mix 2000 4326708 Reactions 10 Pack TaqMan 2X Universal PCR Master Mix 4305719 TaqMan 2X Universal PCR Master Mix No 4324018 AmpErase UNG 200 Reactions TaqMan 2X Universal PCR Master Mix No 4326614 AmpErase UNG 2000 Reactions 10 Pack TaqMan 2X Universal PCR Master Mix 4324020 No AmpErase UNG TagMan PCR Core Reagents Kit 200 Reactions N808 0228 SYBR Green Power SYBR Green PCR Master Mix 1 mL 4368577 reagents 40 reactions Power SYBR Green PCR Master Mix 5 mL 4367659 200 reactions Power SYBR Green PCR Master Mix 4368708 10 x 5 mL 2000 reactions Power SYBR Green PCR Master Mix 50 mL 4367660 2000 reactions SYBR Green PCR Master Mix 1 mL 4344463 40 reactions SYBR Green PCR Master Mix 5 mL 4309155 200 reactions SYBR Green PCR Master Mix 50 mL 4334973 2000 reactions SYBR Green PCR Core Reagents 200 Reactions 4304886 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide D 3 Appendix D Reagent Part Numbers
3. TaqMan reagents separately Kits Part Number TaqMan Exogenous Internal Positive Control Reagents with 4308320 TaqMan 2X Universal PCR Master Mix with VIC dye TaqMan Exogenous Internal Positive Control Reagents 4308323 Note If you are using this kit you will need to purchase one of these TaqMan 2X Universal PCR Master Mix PN 4304437 TaqMan PCR Core Reagents Kit PN N808 0228 IMPORTANT The kits listed above contain TAMRA dye labeled probes Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system The kits may be used with the StepOnePlus system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide D 7 Appendix D Reagent Part Numbers Master Mixes Master Mix Part Number TagMan Gene Expression Master Mix 1 Pack 1 x 5 mL 4369016 200 reactions TagMan Gene Expression Master Mix 10 Pack 10 x 5 mL 4369542 2000 reactions TaqMan 2X Universal PCR Master Mix 200 Reactions 4304437 TaqMan 2X Universal PCR Master Mix 2000 Reactions 4326708 10 Pack TaqMan 2X Universal PCR Master Mix 4305719 TagMan 2X Universal PCR Master Mix No AmpErase UNG 200 4324018 Reactions TaqMan 2X Universal PCR Master Mix No AmpErase UNG 2000 4326614 Reactions 10 Pack TaqMan 2X Universal PCR Maste
4. Reverse Primer GR0746 Figure 3 1 Schematic representation of 1 step RT PCR In 2 step RT PCR you perform two separate reactions one for RT and one for PCR Figure 3 2 2 step RT PCR is useful when detecting multiple transcripts from a single cDNA reaction or when storing a portion of the cDNA for later use When you perform PCR using dUTP as one of the bases you can use AmpErase UNG enzyme to prevent carryover contamination For information about UNG see Using UNG to Minimize Reamplification of Carryover Products on page 2 7 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 3 Quantitation Experiments Primer extended on mRNA 5 3 mRNA RT lt r ED CDNA everse Step i rimer Synthesis of 1st cDNA strand Olige amer random hexamer y 5 CDNA PCR Step Figure 3 2 Schematic representation of 2 step RT PCR Primers Used for For 1 step RT PCR sequence specific reverse primers can be used for cDNA cDNA Synthesis synthesis For 2 step RT PCR the following primers can be used for cDNA synthesis Oligo 76 Random primers Sequence specific reverse primers The choice of primers for reverse transcription is best made after experimentally evaluating all three priming systems For short RNA sequences containing no hairpin loops any of the three priming systems works equally well For longer RNA transcripts or sequences containing hairpin loops consider the
5. Consumable MicroAmp Fast Optical 48 Well Reaction Plate MicroAmp Fast 48 Well Tray MicroAmp 96 Well Support Base MicroAmp Optical 8 Cap Strip MicroAmp Fast 8 Tube Strip MicroAmp Fast Reaction Tube with Cap MicroAmp 48 Well Optical Adhesive Film T O m m gt lt MicroAmp 48 Well Base Adaptor 1 4 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 1 Introduction StepOnePlus The StepOnePlus system supports the consumables listed below These consumables System are for use with both standard and Fast reagents protocols IMPORTANT Use only Fast consumables reaction plates tube strips and tubes with the StepOne and StepOnePlus systems even when performing an experiment with standard reagents Consumable Part Number MicroAmp Fast Optical 96 Well Reaction Plate with 4346906 and Barcode 4366932 MicroAmp Optical Adhesive Film 4360954 and 4311971 e MicroAmp Fast 8 Tube Strip e 4358293 e MicroAmp Optical 8 Cap Strip e 4323032 e MicroAmp Fast Reaction Tube with Cap e 4358297 e MicroAmp 96 Well Tray for VeriFlex Blocks e 4379983 MicroAmp 96 Well Support Base e 4379590 MicroAmp Adhesive Film Applicator e 4333183 e MicroAmp Cap Installing Tool Handle e 4330015 Consumable MicroAmp Fast Optical 96 Well Reaction Plate
6. Distinct clusters and high call rates for unambiguous allelic discrimination Excellent pre and post PCR stability for high throughput setup and analysis e Validated with TaqMan SNP Genotyping Assays e Simplifies assay implementation by using one reagent for all assays TagMan 2X Universal PCR Master Mix with or without AmpErase UNG e Provides optimal performance for TaqMan assays that use CDNA or DNA as a template e Contains components that ensure excellent assay performance e Simplifies assay implementation by using one reagent for all assays Note Genotyping experiments are not supported for Fast or SYBR Green master mixes and protocols Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 4 Genotyping Experiments For guidelines on designing your experiments with Pre Designed Validated assays see page 4 10 Custom Assays Product Attributes Custom TaqMan SNP Genotyping Assays Any possible single nucleotide polymorphism SNP in any organism Detect insertions deletions in dels of up to six bases Detect multiple nucleotide polymorphisms MNPs of up to six bases Convenient single tube format TagMan Genotyping Master Mix Optimized for endpoint fluorescence detection in SNP genotyping applications Distinct clusters and high call rates for unambiguous allelic discrimination Excellent pre and p
7. Regression coefficient calculated from the regression line in the standard curve The slope indicates the PCR amplification efficiency for the assay A slope of 3 32 indicates 100 amplification efficiency See also amplification efficiency EFF and regression line Abbreviation for single nucleotide polymorphism The SNP can consist of a base difference or an insertion or deletion of one base Used in genotyping experiments a PCR reaction that contains primers to amplify the SNP and two probes to detect different alleles In the StepOne software a collection of SNP assays to add to genotyping experiments The SNP assays in the library contain the SNP assay name SNP assay color and for each allele the allele name or base s reporter quencher and allele colors The SNP assays in the library may also contain the assay ID and comments about the SNP assay Type of StepOne and StepOnePlus system calibration in which the system maps the positions of the wells in the sample block s Spatial calibration data are used so that the software can associate increases in fluorescence during a run with specific wells in the reaction plate In the thermal profile a group of one or more steps There are three types of stages holding stage including pre PCR read and post PCR read cycling stage also called amplification stage and melt curve stage Glossary 11 Glossary standalone layout standard standard curve stan
8. Standard dilution series A set of standards containing a range of known quantities The standard dilution series is prepared by serially diluting standards Endogenous control A target or gene that should be expressed at similar levels in all samples you are testing The endogenous control is used to normalize fluorescence signals for the target you are quantifying Housekeeping genes can be used as endogenous controls Replicates The total number of identical reactions containing identical samples components and volumes Negative Controls Wells that contain water or buffer instead of sample template No amplification of the target should occur in negative control wells The comparative Cy AAC method is used to determine the relative target quantity in samples With the comparative C method the StepOne software measures amplification of the target and of the endogenous control in samples and in a reference sample Measurements are normalized using the endogenous control The software determines the relative quantity of target in each sample by comparing normalized target quantity in each sample to normalized target quantity in the reference sample Comparative C4 experiments are commonly used to Compare expression levels of a gene in different tissues Compare expression levels of a gene in a treated sample vs an untreated sample Compare expression levels of wild type alleles vs mutated alleles
9. Allows you to run quantitation experiments on the StepOne and StepOnePlus systems in about 35 min IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system For guidelines on designing your experiments with Inventoried Made to Order assays see page 3 16 3 13 Chapter 3 Quantitation Experiments Custom Assays 3 14 Product Attributes Custom TaqMan Probes and Primers Any species or organism Choice of dye labels quenchers and synthesis scales For use with the Primer Express Software and Applied Biosystems Assay Design Guidelines Primer Express Software Software that designs primers and probes for real time PCR TagMan Gene Expression Master Mix Tailored for precise quantitation by real time PCR for routine and challenging experiments Sensitive detection down to 1 copy of target Multiplex PCR for co amplifying two targets in a single reaction Specificity for differentiation between gene family members Validated with TaqMan Gene Expression Assays Simplifies assay implementation by using one reagent for all assays TaqMan 2X Universal PCR Master Mix with or without AmpErase UNG Provides optimal performance for TaqMan assays that use cDNA or DNA as a template Contains components that ensure excellent assa
10. Quantitation experiments standard curve relative standard curve and comparative C cDNA complementary cDNA RNA or gDNA genomic DNA For quantitation experiments the template type selection affects the run method reaction setup and materials list Genotyping experiments Wet DNA gDNA or cDNA or dry DNA gDNA or cDNA For genotyping experiments the template type selection affects the reaction setup Presence absence experiments DNA For presence absence experiments Applied Biosystems recommends adding DNA templates to the PCR reactions Part of the run method that specifies the temperature time ramp and data collection points for all steps and stages of the StepOne or StepOnePlus instrument run 1 In amplification plots the level of fluorescence above the baseline and within the exponential growth region The threshold can be determined automatically see automatic C4 or can be set manually see manual C 2 In presence absence experiments the level of fluorescence above which the StepOne software assigns a presence call The PCR cycle number at which the fluorescence meets the threshold in the amplification plot See melting temperature Tm Instrument display that you touch to control the StepOne or StepOnePlus instrument Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide unknown unknown IPC wells VeriFlex Technology y interce
11. Real Time e 48 well platform PCR System StepOne system Three color system Applied Biosystems StepOnePlus Real e 96 well platform Time PCR System StepOnePlus system Four color system e VeriFlex sample blocks The StepOne and StepOnePlus systems use fluorescent based polymerase chain reaction PCR reagents to provide Quantitative detection of target nucleic acid sequences targets using real time analysis Qualitative detection of targets using post PCR endpoint analysis Qualitative analysis of the PCR product achieved by melt curve analysis that occurs post PCR About Data The StepOne and StepOnePlus systems collect raw fluorescence data at different Collection points during a PCR depending on the type of run that the instruments perform Run Type Data Collection Point Real time runs Standard curve The instrument collects data following each extension step of the PCR Relative standard curve Comparative C4 AAC Post PCR Genotyping The instrument collects data endpoint runs Presence absence Before the PCR For presence absence experiments data collection before the PCR is optional but recommended e Optional During the PCR The instrument can collect data during the run real time collecting data during the run can be helpful for troubleshooting e After the PCR Regardless of the run type a data collection point or r
12. TaqMan Drug Metabolism Genotyping Assays Protocol 4362038 TaqMan SNP Genotyping Assays Protocol 4332856 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide ix Preface Documents Related to Presence Absence Experiments Document PN DNA Isolation from Fresh and Frozen Blood Tissue Culture Cells and 4343586 Buccal Swabs Protocol NucPrep9 Chemistry Isolation of Genomic DNA from Animal and Plant 4333959 Tissue Protocol PrepMan Ultra Sample Preparation Reagent Protocol 4318925 Documents Related to Relative Standard Curve and Comparative C4 Experiments Document PN Amplification Efficiency of TagMan Gene Expression Assays 127AP05 Application Note Applied Biosystems High Capacity cDNA Reverse Transcription Kits 4375575 Protocol Custom TaqMan Gene Expression Assays Protocol 4334429 Primer Express Software Version 3 0 Getting Started Guide 4362460 TaqMan Gene Expression Assays Protocol 4333458 User Bulletin 2 Relative Quantitation of Gene Expression 4303859 Documents Related to Standard Curve Experiments Document PN Amplification Efficiency of TaqMan Gene Expression Assays 127AP05 Application Note Custom TaqMan Gene Expression Assays Protocol 4334429 Primer Express Software Version 3 0 Getting Started Guide 4362460 TaqMan Gene Expression Assays Protocol 4333458 User Bulletin 2 Rel
13. TaqMan SNP Genotyping Assays and Custom TaqMan SNP Genotyping Assays can be used with Master Mix Part Number TaqMan Genotyping Master Mix 1 Pack 1 x 10 mL 4371355 400 reactions TaqMan Genotyping Master Mix 1 Bulk Pack 1 x 50 mL 4371357 2000 reactions TaqMan 2X Universal PCR Master Mix 200 Reactions 4304437 TaqMan 2X Universal PCR Master Mix 2000 Reactions 4326708 10 Pack TagMan 2X Universal PCR Master Mix 4305719 TaqMan 2X Universal PCR Master Mix No AmpErase 4324018 UNG 200 Reactions TaqMan 2X Universal PCR Master Mix No AmpErase 4326614 UNG 2000 Reactions 10 Pack TaqMan 2X Universal PCR Master Mix No 4324020 AmpErase UNG Note Genotyping experiments are not supported for Fast or SYBR Green master mixes and protocols For More For information on using the TaqMan master mixes refer to the Information TaqMan Genotyping Master Mix Protocol TaqMan Universal PCR Master Mix Protocol Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 4 13 Chapter 4 Genotyping Experiments Design the Experiment Use the StepOne For Applied Biosystems Pre Designed Validated assays use the StepOne software to Software design your genotyping experiments The StepOne software automatically calculates volumes for the Reaction mix components Sample dilutions Note To select a SNP assay in the StepOne software go to the SNP Assays screen in the Design Wizard
14. dye labeled TagMan Endogenous Control Assays are included as Inventoried TaqMan Gene Expression Assays Custom TaqMan Gene Expression Assays For information on the latest available products and specific product uses refer to the Applied Biosystems Web site http www appliedbiosystems com Master Mixes Master Mix Part Number TaqMan Gene Expression Master Mix 1 Pack 1 x 5 mL 4369016 200 reactions TaqMan Gene Expression Master Mix 10 Pack 10 x 5 mL 4369542 2000 reactions TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG 4352042 250 Reactions TaqMan 2X Universal PCR Master Mix 200 Reactions 4304437 TagMan 2X Universal PCR Master Mix 2000 Reactions 4326708 10 Pack TaqMan 2X Universal PCR Master Mix 4305719 TaqMan 2X Universal PCR Master Mix No AmpErase UNG 200 4324018 Reactions TaqMan 2X Universal PCR Master Mix No AmpErase UNG 2000 4326614 Reactions 10 Pack TaqMan 2X Universal PCR Master Mix No AmpErase UNG 4324020 Custom Assays Assays Product Part Number Custom TaqMan Probes and Primers For information on the latest available products and specific product uses refer to the Applied Biosystems Web site http www appliedbiosystems com D 2 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Appendix D Reagent Part Numbers
15. Components The following components are required when setting up PCR reactions for comparative C experiments Sample The sample in which the quantity of the target is unknown Reference sample The sample used as the basis for relative quantitation results For example in a study of drug effects on gene expression an untreated control would be an appropriate reference sample Also called calibrator Endogenous control A target or gene that should be expressed at similar levels in all samples you are testing The endogenous control is used to normalize fluorescence signals for the target you are quantifying Housekeeping genes can be used as endogenous controls Replicates The total number of identical reactions containing identical samples components and volumes Negative Controls Wells that contain water or buffer instead of sample template No amplification of the target should occur in negative control wells Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Comparison of Chapter 3 Quantitation Experiments Consider the following when choosing between standard curve relative standard Quantitation curve and comparative C4 experiments Methods Experiment E uiae ce Type Description Advantage Limitation Standard curve Uses a standard curve to determine the absolute quantity of a target in a sample Typically used for quantifying viral load Allows com
16. MicroAmp 96 Well Tray for VeriFlex Blocks MicroAmp 96 Well Support Base MicroAmp Optical 8 Cap Strip MicroAmp Fast 8 Tube Strip MicroAmp Fast Reaction Tube with Cap Ol moc o u gt gt MicroAmp Optical Adhesive Film Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 1 5 Chapter 1 Introduction Preparing for Experiments General Workflow Before performing experiments on the StepOne and StepOnePlus systems prepare for the experiment as follows 1 Select an experiment type page 1 7 2 Select the reagent type page 1 8 3 Select the assay type page 1 9 Information in This chapter provides general information on the experiment types reagent types This Guide and assay types you can use with the StepOne and StepOnePlus systems Subsequent chapters provide specific information Chapter Description Chapter 2 Reagent Overview e Describes and compares the TaqMan and SYBR Green reagents e Provides information on minimizing DNA contamination Chapter 3 Quantitation e Explains how the experiment type works Experiments Provides a specific workflow for the experiment type Chapter 4 Genotyping Experiments Provides design guidelines for each assay type Chapter 5 Presence Absence Experiments 1 6 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Cha
17. The software uses the baseline and threshold to calculate the threshold cycle Cy See also threshold cycle Cy In the amplification plot a line fit to the fluorescence levels during the initial stages of PCR when there is little change in fluorescence signal Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide baseline corrected normalized reporter ARn blocked IPC calibrator chemistry colocated layout comparative C AAC method Cr custom dye cycle threshold Glossary The magnitude of normalized fluorescence signal generated by the reporter 1 In experiments that contain data from real time PCR the magnitude of normalized fluorescence signal generated by the reporter at each cycle during the PCR amplification In the ARn vs Cycle amplification plot ARn is calculated at each cycle as ARn cycle Rn cycle Rn baseline where Rn normalized reporter 2 In genotyping experiments and presence absence experiments the difference in normalized fluorescence signal generated by the reporter between the pre PCR read and the post PCR read In the allelic discrimination plot genotyping experiments and the presence absence plot presence absence experiments ARn is calculated as ARn Rn post PCR read Rn pre PCR read where Rn normalized reporter See also normalized reporter Rn In presence absence experiments a reaction that contains IPC blocking
18. in a reference sample and in a standard dilution series Measurements are normalized using the endogenous control Data from the standard dilution series are used to generate the standard curve Using the standard curve the software interpolates target quantity in the samples and in the reference sample The software determines the relative quantity of target in each sample by comparing target quantity in each sample to target quantity in the reference sample Relative standard curve experiments are commonly used to Compare expression levels of a gene in different tissues Compare expression levels of a gene in a treated sample vs an untreated sample Compare expression levels of wild type alleles vs mutated alleles Components The following components are required when setting up PCR reactions for relative standard curve experiments Sample The sample in which the quantity of the target is unknown Reference sample The sample used as the basis for relative quantitation results For example in a study of drug effects on gene expression an untreated control would be an appropriate reference sample Also called calibrator Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 3 5 Chapter 3 Quantitation Experiments About Comparative Cz Experiments 3 6 Standard A sample that contains known standard quantities used in quantitation experiments to generate standard curves
19. on page 3 10 You can use other fluorescent based reagents on the StepOne and StepOnePlus systems but note the following when using the StepOne software You must design your experiment using Advanced Setup instead of the Design Wizard For Applied Biosystems TaqMan and SYBR Green reagents the StepOne software automatically calculates reaction volumes in the Reaction Setup screen Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 1 Introduction Select the Assay Type Quantitation and Presence Absence Experiments In the StepOne software you can select the following assay types for quantitation presence absence and genotyping experiments Experiment Type Assay Type See Quantitation nventoried Made to Order below Custom Presence absence Genotyping Pre Designed Validated page 1 10 Custom User Designed t Quantitation experiments include standard curve relative standard curve and comparative C experiments Inventoried Made to Order Assays For quantitation or presence absence experiments select the Inventoried Made to Order assay type in the StepOne software if you are using TaqMan Gene Expression Assays Inventoried Predesigned FAM dye labeled TaqMan MGB minor groove binder probe and primer sets that can be purchased off the shelf The assay mix is available in a single preformulated 20X tube TaqMan Gene Exp
20. singleplex comparison 3 10 MultiScribe reverse transcriptase defined 3 26 N negative control component of experiment 3 5 3 6 5 4 negative controls 4 4 nonspecific product contamination with SYBR Green dye 2 7 0 optimization 3 33 other fluorescent based reagents 1 8 P PCR general practices 8 positive controls 4 4 Pre Designed Validated assay type 0 presence absence experiments Assay Design Guidelines for Custom assay type 5 16 assay types 5 6 components 5 4 defined 5 4 designing 5 16 how they work 5 5 incorporating an IPC 5 5 performing without an IPC 5 4 selecting master mix 5 15 selecting reagents for Custom assay type 3 24 D 3 D 9 TaqMan reagents 2 2 Primer Express Software presence absence experiments 5 16 quantitation experiments 3 21 small amplicons 3 22 SNP assays 1 10 primer limiting multiplex assays 3 10 Index 2 primer matrix defining limits B 1 example of limiting B 2 how used 3 30 primers 1 step RT PCR 3 9 2 step RT PCR 9 default concentrations 3 30 hairpin loops 3 9 summary of design guidelines 3 23 probes available TaqMan MGB probes 2 4 optimizing probe concentration 3 33 summary of design guidelines 3 23 Q quantitation experiments Assay Design Guidelines for Custom assay type 3 21 assay types 3 12 comparative CT 3 6 comparing 3 7 conclusions for Assay Design Guidelines C 1 designing 3 20 3 21 explained 3 4 real time PCR 3 4 relative standard curve 3 5 selecting a quantita
21. 16 TaqMan Gene Expression Assays Product TaqMan Gene Expression Assays are a comprehensive collection of Inventoried Description and Made to Order probe and primer sets that you can use to perform presence absence experiments on human mouse rat Arabidopsis Drosophila C elegans C familiares dog and M mulatta Rhesus genes The assays e Use TagMan reagents to amplify and detect the target in cDNA samples Are designed using an automated design and quality controlled pipeline Inventoried assays are manufactured and placed in inventory Made to Order assays are predesigned and manufactured when ordered e Are designed and optimized to work with an Applied Biosystems TaqMan master mix using universal thermal cycling conditions When possible amplify target cDNA without amplifying genomic DNA m suffix in assay ID by designing probes that cross exon exon junctions Product All TaqMan Gene Expression Assays require Requirements Three components 10 100 ng of cDNA sample converted from RNA per well with all wells in a study having the same amount of cDNA 20X Gene Expression Assay Mix specific for each target Each assay mix consists of two unlabeled PCR primers and a FAM dye labeled TaqMan MGB minor groove binder probe in a preformulated 20X mix 1X final concentrations are 250 nM for the probe and 900 nM for each primer TaqMan Gene Expression Master Mix or TaqMan Universal PCR Mas
22. Bl 25255 L 285 29 E 2 Bg 0204 p 94 6 8 o 1 12 1 2 1 4 Reverse primer POM Figure B 1 Results from Limiting Primer Matrix experiment a Shows how C4 value is affected by variation in forward and reverse primer concentrations Plateau region indicated shows area where C value remains constant b Shows reduction in AR values as primer concentration decreases Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide B 3 Appendix B Primer Limiting in Multiplex PCR B 4 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Assay Design Guidelines About Assay If you are designing your own assays primers and probes Applied Biosystems Design recommends that you follow the Applied Biosystems Assay Design Guidelines The Guidelines Assay Design Guidelines specify that you 1 Design primers and probes using Primer Express Software The Primer Express software uses a set of default parameters to automatically select primer and probe sets 2 Select the appropriate reagents There are several TaqMan and SYBR Green reagents available The reagents you use depend on your assay type 3 Use the recommended thermal cycling conditions Use the thermal cycling conditions recommended for your sample DNA cDNA RNA for 1 step PCR and RNA for 2 step PCR Note Thermal cycling conditions for Fast reagents differ from thermal c
23. C as a result any of the plot group A or B provides optimal performance Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide SYBR Green Reagents Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 3 Quantitation Experiments a Linear view 2300 nd A Plot Group A jr c p d Plot Group B 1300 a gt fo V 8 000 61 Plot Group C 3 000 61 b Log view Plot Group A Plot Group B 1 000 Plot Group C dius ar 1000 E2 AMA Figure 3 4 Primer optimization experimental results showing amplification plots linear and log views of primer combinations Plot group key A Combinations that contain at least 300 nM of forward and reverse primer B Combinations that contain at least 150 nM of forward and reverse primer C Combinations that contain at least 50 nM of forward and reverse primer 1400 E3 Cycle Optimizing primer concentrations is slightly more complex for quantitation assays using SYBR Green reagents You should perform the same primer optimization matrix as for TaqMan reagents however you must include negative controls for SYBR Green reagents The primer concentrations you select should provide a low C4 and high ARn when run against the target template but should not produce nonspecific product formation with negative controls 3 81 Chapter 3 Quantitation Experiments 3
24. DNA sample 20X 40X or 80X SNP Genotyping Assay Mix specific for each polymorphism Each assay consists of sequence specific forward and reverse primers to amplify the SNP of interest and two TaqMan MGB probes One probe labeled with VIC dye detects the Allele 1 sequence one probe labeled with FAM dye detects the Allele 2 sequence TaqMan Genotyping Master Mix or TaqMan 2X Universal PCR Master Mix with or without AmpErase UNG PCR amplification and an endpoint read to obtain results 4 10 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 4 Genotyping Experiments Available Assays TaqMan SNP Genotyping Assays are available as TaqMan Pre Designed SNP Genotyping Assays Over 4 5 million predesigned genome wide assays including 3 5 million human HapMap SNPs 70 000 human cSNPs 160 000 human validated and 10 000 mouse assays Available in small medium and large scale Made to Order that is manufactured at the time of order For More For information on the latest available products and specific product uses refer Information to the Applied Biosystems Web sites http www allsnps com and or http www appliedbiosystems com a On the Home page under TaqMan Products select TaqMan SNP Genotyping Assays b On the SNP Genotyping Assays page under Pre Designed Validated Assays select TaqMan SNP Genotyping Assays For information on preparing
25. Fast Universal PCR Master Mix 2X No 4352042 AmpErase UNG 250 Reactions TaqMan 2X Universal PCR Master Mix 200 4304437 Reactions TaqMan 2X Universal PCR Master Mix 2000 4326708 Reactions 10 Pack TaqMan 2X Universal PCR Master Mix 4305719 TagMan 2X Universal PCR Master Mix No 4324018 AmpErase UNG 200 Reactions TaqMan 2X Universal PCR Master Mix No 4326614 AmpErase UNG 2000 Reactions 10 Pack TaqMan 2X Universal PCR Master Mix 4324020 No AmpErase UNG TagMan PCR Core Reagents Kit 200 Reactions N808 0228 3 24 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 3 Quantitation Experiments Reagent Kit Part Number SYBR Green Power SYBR Green PCR Master Mix 1 mL 4368577 reagents 40 reactions Power SYBR Green PCR Master Mix 5 mL 4367659 200 reactions Power SYBR Green PCR Master Mix 4368708 10 x 5 mL 2000 reactions Power SYBR Green PCR Master Mix 50 mL 4367660 2000 reactions SYBR Green PCR Master Mix 1 mL 4344463 40 reactions SYBR Green PCR Master Mix 5 mL 4309155 200 reactions SYBR Green PCR Master Mix 50 mL 4334973 2000 reactions SYBR Green PCR Core Reagents 200 Reactions 4304886 RNA Quantitation Using 1 Step Reagent Kit Part Number RT PCR TagMan reagents TaqMan One Step RT PCR Master Mix 4309169 Reagents Kit TagMan EZ RT PCR Core Reagents N8
26. Guidelines Select the probe first then design the primers as close as possible to the probe without overlapping the probe amplicons of 50 to 150 basepairs are strongly recommended Keep the G C content in the 30 to 80 range Avoid runs of an identical nucleotide especially guanine where runs of four or more Gs should be avoided When using Primer Express Software the When using Primer Express Software the Tm should be 68 to 70 C Tm should be 58 to 60 C No G on the 5 end The five nucleotides at the 3 end should have no more than two G and or C bases Make TaqMan MGB probes as short as possible without being shorter than 13 nucleotides Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 3 23 Chapter 3 Quantitation Experiments Select the Reagents Several TaqMan and SYBR Green reagents are available for quantitation experiments The reagents you use depend on your target DNA or cDNA below RNA using 1 step RT PCR page 3 25 RNA using 2 step RT PCR page 3 26 Note If you are using SYBR Green reagents Applied Biosystems highly recommends the Power SYBR Green reagents DNA or cDNA Quantitation Reagent Kit Part Number TaqMan reagents TaqMan Gene Expression Master Mix 1 Pack 4369016 1 x 5 mL 200 reactions TagMan Gene Expression Master Mix 10 Pack 4369542 10 x 5 mL 2000 reactions TagMan
27. MSDSs certificates of analysis and other related documents Download PDF documents Obtain information about customer training Download software updates and patches In addition the Support page provides access to worldwide telephone and fax numbers to contact Applied Biosystems Technical Support and Sales facilities IMPORTANT When directed to do so by this guide or when you need to schedule maintenance for your StepOne or StepOnePlus instrument such as annual planned maintenance or temperature verification calibration contact the Applied Biosystems Care Center To obtain a phone number for or to send an email to the center go to http www appliedbiosystems com support contact Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Introduction This chapter covers About the StepOne and StepOnePlus Systems Supported Consumables 21 22 ca rd ob x CY ORO XC AOL ROGER ed Preparing for Experiments coscecacusu sacas ddp Nd ER Select the Experiment Type 2020222222 ees Select the Reagent Type a ceouadces xk uack cea ene kode une Select the Assay Type c coca seeds ond puce Rob pesce eee Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 1 1 Chapter 1 Introduction About the StepOne and StepOnePlus Systems There are two models available for this Real Time PCR System System Features Applied Biosystems StepOne
28. PCR reaction components that consist of primers designed to amplify the target and a TaqMan probe designed to detect amplification of the target The nucleic acid sequence that you want to amplify and detect In the StepOne software a color assigned to a target to identify the target in the plate layout and analysis plots In the StepOne software a collection of targets to add to experiments The targets in the library contain the target name reporter quencher and target color The target in the library may also contain comments about the target Glossary 13 Glossary task temperature plot template thermal profile threshold threshold cycle C4 Tm touchscreen Glossary 14 In the StepOne software the type of reaction performed in the well for the target or SNP assay Available tasks Unknown Negative Control Standard standard curve and relative standard curve experiments Positive control genotyping experiments PC presence absence experiments Blocked IPC presence absence experiments In the StepOne software a display of temperatures for the sample instrument cover i p play np san p and instrument block during the StepOne or StepOnePlus instrument run In the Design Wizard of the StepOne software and in QuickStart for quantitation experiments the type of nucleic acid to add to the PCR reaction The recommended template varies according to experiment type
29. RNA is always greater than the concentration of any target mRNA Therefore in multiplex reactions amplifying both target and rRNA only the concentrations of the rRNA primers need to be limited To define limiting primer concentrations run a matrix of forward and reverse primer concentrations using the value of the minimum initial template The goal is to identify primer concentrations that reduce the ARn value of the assay without affecting the C value The table below illustrates a recommended matrix of forward and reverse primers varying in concentration from 20 to 100 nM Note Although following all design criteria does facilitate the ability to identify limiting primer concentrations it may not be possible for all assays If a limiting primer matrix experiment does not enable the identification of primer limiting concentrations it will be necessary to redesign at least one primer or run the reactions in separate tubes Forward 100 nM 100 nM 100 nM 100 nM 100 nM Reverse 100 nM 80 nM 60 nM 40 nM 20 nM Forward 80 nM 80 nM 80 nM 80 nM 80 nM Reverse 100 nM 80 nM 60 nM 40 nM 20 nM Forward 60 nM 60 nM 60 nM 60 nM 60 nM Reverse 100 nM 80 nM 60 nM 40 nM 20 nM Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide B 1 Appendix B Primer Limiting in Multiplex PCR Forward 100 nM 100 nM 100 nM 100 nM 100 nM Reverse 100 nM 80 nM 60 nM 40 nM 20 nM Forward 40 nM 40 n
30. Real Time PCR Systems Reagent Guide Appendix A Appendix B Formulas Standard Deviation Calculation Using the Standard Curve Method A 2 Standard Deviation Calculation Using the Comparative Method A 5 Formula for Comparative Cz AAC Experiments eee A 7 Primer Limiting in Multiplex PCR Appendix C Assay Design Guidelines Appendix D Bibliography Glossary Index Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Reagent Part Numbers Quantitation Experiments 0 Ren D 2 Inventoried Made to Order Assays 0000022 D 2 Gustom ASSays Re EE IET D 2 Genotyping Experiments 20022 mn D 5 Pre Designed Validated Assays 20002200 D 5 Gustom ASSays anne Sete rage ee Rey we FULUCEIUsrsrvrru y Fun D 5 Presence Absence Experiments 00 D 7 Inventoried Made to Order Assays 000000222 D 7 G stomAssayscsozci I X DELI Lite Vb eig m I et hae D 8 vi Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Preface How to Use This Guide About the System The guides listed below are shipped with the Applied Biosystems StepOne and Documentation StepOnePlus Real Time PCR Systems StepOne and StepOnePlus systems StepOnePlus Real Time PCR Systems Reagent Guide StepOne and StepOnePlus systems including e An introduction to TaqMan and
31. SYBR Green reagents system dye TagMan reagents target target color Target Library Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Glossary A component of the thermal profile For each step in the thermal profile you can set the ramp rate ramp increment for melt curve steps hold temperature hold time duration and you can turn data collection on or off for the ramp or the hold parts of the step For cycling stages a step is also defined by the AutoDelta status With StepOnePlus systems which contain the VeriFlex blocks each step contains 6 temperatures 1 for each VeriFlex block PCR reaction components that consist of two primers designed to amplify the target and SYBR Green dye to detect double stranded DNA Dye supplied by Applied Biosystems and precalibrated on the StepOne or StepOnePlus system Before you use system dyes in your experiments make sure the system dye calibration is current in the Instrument Maintenance Manager System dyes on the StepOne system FAM dye JOE dye ROX dye SYBR Green dye VIC dye System dyes on the StepOnePlus system FAM dye JOE dye NED dye ROX dye SYBR Green dye TAMRA dye VIC dye IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system
32. StepOne software page 3 20 TaqMan Gene Expression Assays Product TaqMan Gene Expression Assays are a comprehensive collection of Inventoried Description and Made to Order probe and primer sets for performing quantitation experiments on human mouse rat Arabidopsis Drosophila C elegans C familiares dog and M mulatta Rhesus genes The assays e Use TaqMan reagents to amplify and detect the target in cDNA samples Are designed using an automated design and quality controlled pipeline Inventoried assays are manufactured and placed in inventory Made to Order assays are predesigned and manufactured when ordered e Are designed and optimized to work with an Applied Biosystems TaqMan master mix using universal thermal cycling conditions When possible amplify target cDNA without amplifying genomic DNA m suffix in assay ID by designing probes that cross exon exon junctions Product All TaqMan Gene Expression Assays require Requirements Three components 1 to 100 ng of cDNA sample converted from RNA per well with all wells in a study having the same amount of cDNA 20X Gene Expression Assay Mix specific for each target Each assay mix consists of two unlabeled PCR primers and a FAM dye labeled TaqMan MGB minor groove binder probe in a preformulated 20X mix 1X final concentrations are 250 nM for the probe and 900 nM for each primer TaqMan Gene Expression Master Mix TaqMan Univers
33. Temperatures Initial Steps PCR 40 Cycles AmpliTaq Gold Reverse DNA Polymerase Transcription Melt Anneal Extend Activation HOLD HOLD 40 CYCLES 30 min 6 48 C 10 min 6 95 C 15 sec 6 95 C 1 min 6 60 C Note These conditions do not apply to the TaqMan EZ RT PCR Kit See the TaqMan EZ RT PCR Kit Protocol for the appropriate conditions Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide RNA Quantitation Using 2 Step RT PCR Chapter 3 Quantitation Experiments For the High Capacity cDNA Reverse Transcription Kit follow these conditions Step Times and Temperatures Step 1 Step 2 1 RT Step HOLD HOLD 10 min 6 25 C 120 min 6 37 C After reverse transcribing the RNA into cDNA RT step samples can be stored or used for the subsequent PCR step described below IMPORTANT For most applications and when large amounts of cDNA are required Applied Biosystems recommends 120 minutes at 37 C for reverse transcription to achieve optimal conversion For TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG follow these conditions Step Times and Temperatures Initial Steps PCR 40 Cycles AmpliTaq AmpErase D 2 PCR Step UNG Er DNA Melt t Activation o ymerase xen Activation HOLD HOLD CYCLE 2min 50 C 20sec 95 C 1 sec 95 C 20 sec 60 C Required only if Am
34. The c mycy value is determined by dividing the average c myc value by the average GAPDH value The standard deviation of the quotient is calculated from the standard deviations of the c myc and GAPDH values See Formula on page A 4 The calculation of c mycy relative to brain involves division by the reference sample value This is division by an arbitrary constant so the cv of this result is the same as the cv for c mycy Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide A 3 Appendix A Formulas Formula A 4 The c mycy value is determined by dividing the average c myc value by the average GAPDH value The standard deviation of the quotient is calculated from the standard deviations of the c myc and GAPDH values using the following formula 2 2 2 cv fev tcv where stddev s c 2 X meanvalue Using the brain sample from table on page A 2 as an example _ 4 cvi 0 039 and cy 0 034 27 4 0 K Gee c om eq 0P since lt I Le cv s cv X s 0 12 0 07 s 0 008 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Appendix A Formulas Standard Deviation Calculation Using the Comparative Method Example The C data used to determine the amounts of c myc and GAPDH mRNA shown in the table on page A 2 are used to illustrate the AAC calculation The table below
35. Time PCR Systems Reagent Guide 2 5 Chapter 2 Reagent Overview Selecting the Appropriate Reagent Type The TaqMan and SYBR Green reagents can be used for the experiment types listed below Experiment Type Reagent Type Quantitation Genotyping Jesu Chapter 3 Chapter 4 Chapter 5 SYBR Green reagents Yes Not Not recommended recommended TaqMan reagents Yes Yes Yes t Includes standard curve relative standard curve and comparative C experiments IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used asa reporter or quencher with the StepOnePlus system Considerations for Quantitation Experiments Quantitation experiments can be performed with either TagMan or SYBR Green reagents Consider the following when choosing between the two reagent types Reagent Type Description Advantage Limitation TaqMan TaqMan reagents use a e Provides increased Requires synthesis of a unique reagents fluorogenic probe to enable specificity with the addition fluorogenic probe detection of a specific PCR product as it accumulates during PCR cycles of a fluorogenic probe Provides multiplex capability Includes preformulated assays optimized to run under universal thermal cycling conditions Can be used for either 1 or 2 step RT PCR SYBR Green SYBR Green reagents use Is economical
36. a a Ae inie A E ar O Eu Aaa ig dudes TaqMan Reagents accen Loa ut too o s NR B t capt Cede rus SYBR Green Reagents xoc edes tad en eec gelten d eol tad Selecting the Appropriate Reagent Type 5 Minimizing DNA Contaminants slseeeeeee nh Quantitation Experiments Section 3 1 About Quantitation Experiments eee eee eee NIME Select a Quantitation Method Select 1 Step or 2 Step RT PCR 0 ellen Select Singleplex or Multiplex PCR seen Select the Reagent Type 22022 meh Select the Assay Type 222 Section 3 2 Design Guidelines elle Inventoried Made to Order Assays 0 cece eee TaqMan Gene Expression Assays 2222222222 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide lii Chapter 4 Chapter 5 Custom TagMan Gene Expression 88588 77777 7 3 18 Select the Master Mix 3 19 Design the Experiment 3 20 GUstomrASSays eii axle RU ERL UNUS dur LAE EE px ERE 3 21 Design Primers and Probes Using Primer Express Software 3 21 Select the ReagentS isse odd Re RR ERR RUEGG MERE Eee ees 3 24 Use the Recommended Thermal Cycling Conditions 3 27 Optimize Primer Concentrations 00 3 30 Optimize the Probe Concentration 00 3 33 For More Information 0 000 ee eee 3 35 Genotyping Experiments Section 4 1 About Genotyping Experiments 0 4 3 OVGEIVIOW 22 sso B ects
37. automatically calculates volumes for the Reaction mix components Controls and samples Sample dilutions Note To select the Inventoried Made to Order assay type in the StepOne software go to the Reaction Setup screen in either the Design Wizard or Advanced Setup then select Inventoried Made to Order from the Assay Type dropdown menu For information on designing and performing presence absence experiments on the StepOne and StepOnePlus systems refer to the Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Getting Started Guide for Presence Absence Experiments Custom Assays Workflow If you select the Custom assay type in the StepOne software for a presence absence experiment that is you are designing your own primers and probes Applied Biosystems recommends that you follow the workflow for the Applied Biosystems Assay Design Guidelines 1 Design primers and probes using Primer Express Software page 3 21 2 Select the appropriate reagents page 3 24 IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system Presence absence experiments are not supported for Fast or SYBR Green master mixes and protocols 3 Use the recommended thermal cycling conditions page 3 27 4 Begin with default primer and probe concentrations If needed optimize the pri
38. during the amplification stage are displayed in an amplification plot and the data can be used for troubleshooting See also cycling stage In the StepOne and StepOnePlus systems a PCR reaction mix that contains primers to amplify a target and a reagent to detect the amplified target Identifier assigned by Applied Biosystems to TaqMan Gene Expression Assays and TaqMan SNP Genotyping Assays Data file on a CD shipped with each assay order The file name includes the number from the barcode on the plate The information in the AIF is provided in a tab delimited format PCR reaction component in Applied Biosystems TaqMan Gene Expression Assays and TaqMan SNP Genotyping Assays The assay mix contains primers designed to amplify a target and a TaqMan probe designed to detect amplification of the target In the run method a setting to increase or decrease the temperature and or time for a step with each subsequent cycle in a cycling stage When AutoDelta is enabled for a cycling stage the settings are indicated by an icon in the thermal profile AutoDelta on A AutoDelta off A An analysis setting in which the software calculates the baseline start and end values for the amplification plot You can apply the automatic baseline setting to specific wells in the reaction plate See also baseline An analysis setting in which the software calculates the baseline start and end values and the threshold in the amplification plot
39. er ordin 0 re e pun solae eurer e e bud Rex eae Mea He 4 4 Selectilie Assay Type esI RIT EUDEVESScASENESUELMUL MERGE GE 4 6 Section 4 2 Design Guidelines 0 00 cece eee 4 9 Pre Designed Validated Assays 00 cece eres 4 10 TagMan SNP Genotyping Assays 0 0 cece cece eee ee eens 4 10 TaqMan Drug Metabolism Genotyping Assays 2 00 4 11 Pre Developed TaqMan Assay Reagents for Allelic Discrimination 4 12 Select the Master Mix 7222 4 13 Design the Experiment sorceres a y ea E EE Ey ees 4 14 Custom ASSayS td Rd arn 5 hh din e m ot ete vb ea tot de umet gts 4 14 Custom TaqMan SNP Genotyping Assays 2220202222 4 14 Select the Master Mix 0 00 e eR e eh 4 16 Design the Experiment 002 lene 4 16 Presence Absence Experiments Section 5 1 About Presence Absence Experiments 5 3 OVGIVIOW uie Sint ee ed dne x Mon E ES OAI PE UN E 5 4 Select the Assay Type e etp reaa ai a i a aa aa A aa a Rn 5 6 Section 5 2 Design Guidelines 0 cece ee eee eens 5 9 Inventoried Made to Order Assays eee eee 5 10 TaqMan Gene Expression Assays 2 02 5 10 Custom TaqMan Gene Expression Assays lies eese 5 12 TaqMan Exogenous Internal Positive Control Reagents 5 13 Select the Master Mix 00 cece 5 15 Design the Experiment 0 0c cece eee ees 5 16 5 16 Applied Biosystems StepOne and StepOnePlus
40. following guidelines Primers Selection Guidelines Oligo d T 4s e Use to reverse transcribe only eukaryotic mRNAs and retroviruses with poly A tails e Avoid long mRNA transcripts or amplicons greater than 2 kilobases upstream from the poly A site Random primers Try first for use with long reverse transcripts or reverse transcripts containing hairpin loops e Use to transcribe all RNA rRNA mRNA and tRNA Sequence specific e Use to reverse transcribe RNA containing complementary reverse primers sequences only e Use in 1 step RT PCR Comparison of RT PCR Methods Method Primers for cDNA Synthesis Comments 1 step Sequence specific reverse primer Requires single reaction mix Ben AmpErase UNG cannot be used 2 step Random hexamers cDNA can be stored for later use RT PCR Oligo d T AmpErase UNG can be used Sequence specific reverse primers Requires two reaction mixes Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 3 9 Chapter 3 Quantitation Experiments Select Singleplex or Multiplex PCR About Multiplex PCR You can perform a PCR reaction using either Singleplex PCR In singleplex PCR a single primer set is present in the reaction tube or well Only one target or endogenous control can be amplified per reaction or Multiplex PCR In multiplex PCR two or more primer sets are present in the reaction tube or well Ea
41. level of fluorescence rather than the final quantity of PCR product accumulated after a fixed number of cycles An amplification plot graphically displays the fluorescence detected over the number of cycles that were performed In the initial cycles of PCR there is no significant change in fluorescence signal This predefined range of PCR cycles is called the baseline First the software generates a baseline subtracted amplification plot by calculating a mathematical trend of the normalized fluorescent reporter signal Rn values corresponding to the baseline cycles Then an algorithm searches for the point on the amplification plot at which the baseline corrected normalized fluorescent reporter signal delta Rn ARn value crosses a set threshold The cycle at which the ARn value crosses the threshold is defined as the Cp Before performing quantitation experiments on the Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems prepare for the experiment as follows 1 Select a quantitation method page 3 5 Select 1 or 2 step RT PCR page 3 8 Select singleplex or multiplex PCR reactions page 3 10 Select the reagent type page 3 12 Select the assay type page 3 12 SA dec oo Review the design guidelines for the assay type you selected Section 3 2 on page 3 15 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 3 Quantitation Experiments Select a Quantitat
42. no probe Binds nonspecifically to all reagents SYBR Green dye a double needed double stranded DNA stranded DNA binding dye to Allows for melt curve sequences To avoid false detect PCR products as they analysis to measure the Tm positive signals check for accumulate during PCR of all PCR products nonspecific product formation Can be used for either 1 or Md ee Cuive or gel 2 step RT PCR 2 6 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 2 Reagent Overview Minimizing DNA Contaminants The DNA amplification capability of the PCR process makes special laboratory practices necessary when you perform experiments using TaqMan or SYBR Green reagents Potential contamination can be introduced by samples with high DNA concentrations either from the DNA template controls or from PCR carryover In addition due to the nonspecific nature of the SYBR Green I dye any double stranded DNA will be detected When using SYBR Green reagents check for nonspecific product formation by using melt curve or gel analysis Take care to avoid contamination with target DNA Gene expression assays that span exon exon junctions minimize the effect of gDNA genomic DNA contaminants Using UNG to AmpErase uracil N glycosylase UNG is a 26 kDa recombinant enzyme encoded Minimize by the Escherichia coli uracil N glycosylase gene This gene has been inserted into Reamplification an coli ho
43. of collecting fluorescence data during PCR Data from the real time PCR are used to calculate results for quantitation experiments or to troubleshoot results for genotyping or presence absence experiments In relative standard curve and comparative Cy AAC experiments the sample used as the basis for relative quantitation results Also called the calibrator Identifies the reference SNP refSNP cluster ID Generated by the Single Nucleotide Polymorphism Database of Nucleotide Sequence Variation dbSNP at the National Center for Biotechnology Information NCBI The refSNP ID can be used to search the Applied Biosystems Store for an Applied Biosystems SNP Genotyping Assay Also called an rs number Values calculated from the regression line in standard curves including the R value slope and y intercept You can use the regression coefficients to evaluate the quality of results from the standards See also standard curve In standard curve and relative standard curve experiments the best fit line from the standard curve Regression line formula C m log Qty b where m is the slope b is the y intercept and Qty is the standard quantity See also regression coefficients An action that the software performs during analysis to remove one or more wells from further analysis if a specific flag is applied to the well Rejected wells contain results calculated up to the point of rejection Applied Biosystems StepOne and Step
44. on purchasing licenses may be obtained by contacting the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA NOTICE TO PURCHASER PLEASE REFER TO THE USER S GUIDE OR PRODUCT INSERT OF THE REAGENTS NAMED HEREIN FOR LIMITED LABEL LICENSE OR DISCLAIMER INFORMATION TRADEMARKS Applied Biosystems AB Design MicroAmp Primer Express and VIC are registered trademarks and FAM JOE MicroAmp MultiScribe NED ROX StepOne StepOnePlus TAMRA TET and VeriFlex are trademarks of Applied Biosystems or its subsidiaries in the U S and or certain other countries AmpErase AmpliTaq Gold and TaqMan are registered trademarks of Roche Molecular Systems Inc SYBR is a registered trademark of Molecular Probes Inc All other trademarks are the sole property of their respective owners Part Number 4379704 Rev D 12 2008 Contents Preface Chapter 1 Chapter 2 Chapter 3 How to Use This Guide RR RR rta How to Obtain More Information eee How to Obtain Support Ax cx e xem E EE ENS Introduction About the StepOne and StepOnePlus Systems 0 Supported Consumables eesseell en Preparing for Experiments 0000 nen Select the Experiment Type Select the Reagent Type 0000s Select the Assay Type o serrr errai iaa E A a E n Reagent Overview OVervVIGW i eee n o
45. shows the average C4 results for the human brain kidney liver and lung samples and how these Cys are manipulated to determine AC AAC and the relative amount of c myc mRNA The results are comparable to the relative c myc levels determined using the standard curve method Tissue c myc GAPDH ACy AAC c myCy Average C Average C4 c myc GAPDH ACT ACT Brain Rel to Brain Brain 5 23 6340 09 6 86 0 17 0 00 0 17 1 0 0 9 1 1 Kidney 27 03 0 06 220 8 00 2 50x0 10 5 6 5 3 6 0 Liver 26 25 0 07 24 60 0 07 1 65 0 10 5 21 0 10 37 0 84 5 39 7 Lung 25 8340 07 23 01 0 07 2 81 0 10 4 05 0 10 16 5 15 4 17 7 t The AC value is determined by subtracting the average GAPDH C value from the average c myc C value The standard deviation of the difference is calculated from the standard deviations of the c myc and GAPDH values See Formula on page A 6 The calculation of AAC involves subtraction by the AC reference sample value This is subtraction of an arbitrary constant so the standard deviation of AAC is the same as the standard deviation of the AC value The range given for c myc relative to brain is determined by evaluating the expression 2 44Cr with AAC s and AAC s where s the standard deviation of the AAC value Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide A 5 Appendix A Formulas A 6 Formula The AC value is determined by subtracting the average
46. this assumption by checking for nonspecific product formation with either melt curve or gel analysis Most TaqMan assays should enable detection and accurate quantitation to lt 50 copies of a target with even greater sensitivity possible Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide C 1 Appendix C Assay Design Guidelines SYBR Green assays are capable of similar performance however nonspecific product formation can potentially increase the minimum detection limit Conclusions for In general the following conclusion can be made when you use the Assay Design Genotyping Guidelines for genotyping experiments Experiments You can use 900 nM primers a 200 nM probe and 1 to 20 ng of genomic DNA to achieve reproducible and sensitive assay results C 2 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Reagent Part Numbers This appendix covers Quantitation EXDODIBIGBER 24 ide take V RR ORDRE EPA eae Roe desc Res D 2 Genotepiip Ex pennies cuc exa XO ODE GRO GEN eer aes D 5 Presence Absence Experiments ias ada cers ER RR RR XU AC ARS ORE dE D 7 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide D 1 Appendix D Reagent Part Numbers Quantitation Experiments Inventoried Made to Order Assays Assays Product Part Number TaqMan Gene Expression Assays TagMan Endogenous Control Assays Note FAM
47. using the StepOne and StepOnePlus systems Quantitation standard curve Quantitation relative standard curve e Quantitation comparative Cy Melt curve Genotyping Presence absence Entered during experiment setup the name that is used to identify the experiment Experiment names cannot exceed 100 characters and cannot include any of the following characters forward slash backslash V greater than sign gt less than sign asterisk question mark quotation mark vertical line colon or semicolon The type of experiment you are performing using the StepOne or StepOnePlus system Standard curve Comparative Cr AAC Relative standard curve Melt curve not available in the Design Wizard Genotyping Presence absence The experiment type you select affects the setup run and analysis Oligonucleotide that flanks the 5 end of the amplicon The reverse primer and the forward primer are used together in PCR reactions to amplify the target In the thermal profile a stage that includes one or more steps You can add a holding stage to the thermal profile to activate enzymes to inactivate enzymes or to incubate a reaction A gene that is involved in basic cellular functions and is constitutively expressed Housekeeping genes can be used as endogenous controls See also endogenous control In presence absence experiments a short synthetic DNA temp
48. 08 0236 Note Use the TagMan EZ RT PCR Core Reagents when a high temperature RT step is required SYBR Green Power SYBR Green RT PCR Reagents Kit 4368711 reagents SYBR Green RT PCR Reagents 4310179 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 3 25 Chapter 3 Quantitation Experiments RNA Quantitation Using 2 Step Reagent Step Kit Part Number RT PCR TaqMan PCR step only TaqMan Gene Expression Master 4369016 reagents Mix 1 Pack 1 x 5 mL 200 reactions TaqMan Gene Expression Master 4369542 Mix 10 Pack 10 x 5 mL 2000 reactions TagMan 2X Universal PCR Master 4304437 Mix TagMan Fast Universal PCR Master 4352042 Mix 2X No AmpErase UNG RT step only High Capacity cDNA Reverse 4374966 Transcription Kit TagMan Reverse Transcription N808 234 Reagents Both RT and TagMan Gold RT PCR kit N808 232 PCR steps SYBR Green PCR step only Power SYBR Green PCR Master 4367659 reagents Mix SYBR Green PCR Master Mix 4309155 RT step only High Capacity cDNA Reverse 4374966 Transcription Kit TagMan Reverse Transcription N808 234 Reagents Both RT and Power SYBR Green RT PCR 4368711 PCR steps Reagents Kit SYBR Green RT PCR Reagents 4310179 About AmpliTaq The use of the hot start enzyme AmpliTaq Gold DNA Polymerase is an integral Gold DNA part of Applied Biosystems Assay Design Guidelines for both TaqMan and SYBR Po
49. 25 2657 2660 Forster V T 1948 Zwischenmolekulare Energiewanderung und Fluoreszenz Ann Physics Leipzig 2 55 75 Higuchi R Dollinger G Walsh P S and Griffith R 1992 Simultaneous amplification and detection of specific DNA sequences Biotechnology 10 413 417 Higuchi R Fockler C Dollinger G and Watson R 1993 Kinetic PCR Real time monitoring of DNA amplification reactions Biotechnology 11 1026 1030 Kutyavin I V Lukhtanov E A Gamper H B and Meyer R B 1997 Oligonucleotides with conjugated dihydropyrroloindole tripeptides base composition and backbone effects on hybridization Nucleic Acids Res 25 3718 3723 Kwok S and Higuchi R 1989 Avoiding false positives with PCR Nature 339 237 238 Livak K J and Schmittgen T D 2001 Analysis of Relative Gene Expression Data Using Real Time Quantitative PCR and the 2 T Method Methods 25 402 408 Longo M C Berninger M S and Hartley J L 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gene 93 125 128 Saiki R K Scharf S Faloona F et al 1985 Enzymatic amplification of B globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia Science 230 1350 1354 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Bibliography 1 Bibliography Bibliography 2 Applied Biosystems StepOne and StepOnePlus R
50. 32 Melt curves or gel analysis can be extremely useful when you select optimal primer concentrations for quantitation assays using SYBR Green reagents Figure 3 5 on page 3 32 shows the results from a primer optimization matrix at primer concentrations of 900 nM forward and reverse primers Figure 3 5a shows strong amplification of the negative control wells which indicates that significant nonspecific amplification 1s occurring Figure 3 5b shows that the melting temperature of the product generated in the absence of template 18 lower than the melting temperature of the specific product generated with template indicating that significant nonspecific amplification is occurring The results shown in Figure 3 5 are typical of primer dimer formation These results indicate that lower primer concentrations may provide more optimal results Additionally you can redesign another set of primers to the target of interest a Amplification plot linear view Im Target Amplification NC nonspecific amplification 1 5 10 15 20 25 3 35 4i Cycle Number b Melt curve analysis No Template Target Amplification non specific amplification j F l 4 Target 54 NG Amplification gt lt non specific S 3 amplification j D g j NE i l Temperature C Figure 3 5 Amplification data using SYBR Green reagents a Amplification plot linear
51. Applied Reagent Guide Biosystems Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide KS peeps erie Copyright 2008 Applied Biosystems All rights reserved Information in this document is subject to change without notice Applied Biosystems assumes no responsibility for any errors that may appear in this document APPLIED BIOSYSTEMS DISCLAIMS ALL WARRANTIES WITH RESPECT TO THIS DOCUMENT EXPRESSED OR IMPLIED INCLUDING BUT NOT LIMITED TO THOSE OF MERCHANTABILITY OR FITNESS FOR A PARTICULAR PURPOSE IN NO EVENT SHALL APPLIED BIOSYSTEMS BE LIABLE WHETHER IN CONTRACT TORT WARRANTY OR UNDER ANY STATUTE OR ON ANY OTHER BASIS FOR SPECIAL INCIDENTAL INDIRECT PUNITIVE MULTIPLE OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT INCLUDING BUT NOT LIMITED TO THE USE THEREOF For Research Use Only Not for use in diagnostic procedures NOTICE TO PURCHASER Label License The StepOne and StepOnePlus Real Time PCR Systems are covered by one or more of US Patents Nos 5 475 610 5 602 756 6 703 236 6 814 934 7 238 517 7 423 750 and corresponding claims in their non US counterparts owned by Applied Biosystems Inc No right is conveyed expressly by implication or by estoppel under any other patent claim such as claims to apparatus reagents kits or methods such as 5 nuclease methods Further information
52. Assays Protocol Submission Guidelines For information on preparing PCR reactions using the Custom TaqMan Gene Expression Assays refer to the Custom TagMan Gene Expression Assays Protocol TaqMan Exogenous Internal Positive Control Reagents Product The Applied Biosystems TaqMan Exogenous Internal Positive Control Reagents Description contain An internal positive control IPC with predesigned primers and probe PC DNA template PC blocking control The reagents are designed to Distinguish types of negative results A negative call for the target and positive call for the IPC indicates that no target is present A negative call for the target and negative call for the IPC suggests PCR inhibition Avoid amplification of endogenous controls Permit co amplification of the IPC and the target without compromising amplification of the target Detect the IPC using a VIC dye labeled probe Detect the target using a FAM dye labeled probe Work with the TaqMan 2X Universal PCR Master Mix with or without AmpErase UNG using universal thermal cycling conditions Product The TaqMan Exogenous Internal Positive Control Reagents kits require the Requirements following components DNA sample TaqMan assay for your target of interest TagMan 2X Universal PCR Master Mix with or without AmpErase UNG Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 5 13 Chap
53. CR Master Mix No AmpErase UNG 2000 4326614 Reactions 10 Pack TaqMan 2X Universal PCR Master Mix No AmpErase 4324020 UNG Note Genotyping experiments are not supported for Fast or SYBR Green master mixes and protocols For information on using the TaqMan master mixes refer to the TaqMan Genotyping Master Mix Protocol TaqMan Universal PCR Master Mix Protocol Design the Experiment Use the StepOne Software For More Information 4 16 For Applied Biosystems Custom assays use the StepOne software to design your genotyping experiments The StepOne software automatically calculates volumes for the Reaction mix components Sample dilutions Note To select a SNP assay in the StepOne software go to the SNP Assays screen in the Design Wizard or to the Plate Setup screen in Advanced Setup In the SNP Assays screen or Plate Setup screen you can select an assay from the library or create a new assay For information on designing and performing genotyping experiments on the StepOne and StepOnePlus systems refer to Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Getting Started Guide for Genotyping Experiments Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Presence Absence Experiments 5 This chapter covers Section 5 1 About Presence Absence Section 5 2 Design Guidelines 2i sicca ocko RC CRGO AR ECCO EROR A RUE Applied Bi
54. GAPDH C value from the average c myc Cy value The standard deviation of the difference is calculated from the standard deviations of the c myc and GAPDH values using the following formula wo Au 2 s where S std dev Using the brain sample from the table on page A 5 as an example and s 4 0 15 0 09 7 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Appendix A Formulas Formula for Comparative C4 AAC Experiments Formula The quantity of target normalized to an endogenous control and relative to a reference sample is calculated by 2 AACT Derivation of the The equation that describes the exponential amplification of PCR is Formula X X x 1 Ey where number of target molecules at cycle n initial number of target molecules E efficiency of target amplification n number of cycles X initial number of target molecules The threshold cycle C4 indicates the fractional cycle number at which the quantity of amplified target reaches a specified threshold Thus Xr XO Ky where x threshold number of target molecules e Cy x threshold cycle for target amplification constant A similar equation for the endogenous control reaction is Rr RQOX 14 E5 7 Kp where e R threshold number of reference molecules R initial number of reference molecules Ep efficiency of reference amplificat
55. Guide 4 11 Chapter 4 Genotyping Experiments PCR amplification and an endpoint read to obtain results For More For information on the latest available products and specific product uses refer Information to the Applied Biosystems Web sites http www allsnps com and or http www appliedbiosystems com a On the Home page under TaqMan Products select TaqMan SNP Genotyping Assays b On the SNP Genotyping Assays page under Pre Designed Validated Assays select TaqMan Drug Metabolism Genotyping Assays For information on preparing PCR reactions using the TaqMan SNP Genotyping Assays refer to the TagMan Drug Metabolism Genotyping Assays Protocol Pre Developed TaqMan Assay Reagents for Allelic Discrimination Product Pre Developed TaqMan Assay Reagents for Allelic Discrimination TaqMan Description PDARs for AD are Inventoried assays optimized for the discrimination of specific alleles The assays Use TaqMan reagents to amplify and detect specific polymorphisms in purified genomic DNA samples e Are designed and optimized to work with an Applied Biosystems TaqMan master mix using universal thermal cycling conditions Product TaqMan PDARs for AD require three components Requirements 2to 20 ng of purified genomic DNA sample 10 Allelic Discrimination Assay Mix specific for each polymorphism Each assay consists of sequence specific forward and reverse primers to amplify the polymorphic seque
56. IVa PE 00 4 16 Design the Experiment ioosc zs tru setta add udo wa RES ERE d as 4 16 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 4 9 Chapter 4 Genotyping Experiments Pre Designed Validated Assays Workflow Ifyou are designing a genotyping experiment using Applied Biosystems Pre Designed Validated assays Applied Biosystems recommends that you follow the workflow below 1 Select the assay e TagMan SNP Genotyping Assays below TaqMan Drug Metabolism Genotyping Assays page 4 11 TaqMan Pre Developed Assays Reagents for Allelic Discrimination page 4 12 2 Select the master mix page 4 13 3 Design the experiment using the StepOne software page 4 14 TaqMan SNP Genotyping Assays Product TaqMan SNP Genotyping Assays are a comprehensive collection of primer and Description probe sets for genotyping single nucleotide polymorphisms SNPs for human studies The assays Use TaqMan reagents to amplify and detect specific SNP alleles in purified genomic DNA samples Are designed using Applied Biosystems bioinformatics pipeline and software as well as genomic information from Celera Genomics and public databases Are designed and optimized to work with an Applied Biosystems TaqMan master mix using universal thermal cycling conditions Product All TaqMan SNP Genotyping Assays require Requirements Three components to 20 ng of purified genomic
57. M 40 nM 40 nM 40 nM Reverse 100 nM 80 nM 60 nM 40 nM 20 nM Forward 20 nM 20 nM 20 nM 20 nM 20 nM Reverse 100 nM 80 nM 60 nM 40 nM 20 nM Example The results of a limiting primer matrix experiment are shown in Figure B 1 on page B 3 Figure B 1a shows that only when lowering the primer concentrations below approximately 50 nM is the C4 value significantly affected The plateau area shows the region in which suitable primer limiting concentrations can be found In this area the C4 and therefore the corresponding quantitation value is unchanged whereas the ARn value and corresponding product yield are significantly reduced Figure B 1b shows the corresponding relationship between primer concentrations and ARn The figure demonstrates that lower product yields can be achieved by decreasing forward and reverse primer concentrations For this example an appropriate selection of primer limiting concentrations would be at least 50 nM forward and reverse primer Probe concentration should be kept at an optimal level even when an assay is primer limited to ensure that the signal produced is large enough for accurate multicomponenting by the software B 2 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Appendix B Primer Limiting in Multiplex PCR Reverse primer nM Forward primer nM 100 go 60 al Forward primer nM 25255 25 3 26 265 D 265 27 B 5 O 275 28
58. OLYMERIZATION STRAND DISPLACEMENT CLEAVAGE POLYMERIZATION COMPLETED gt w 9 0 o e mode REVERSE PRIMER E 9 Step 1 A reporter RF and Step 2 When both labels Step 3 During each Step 4 Once separated a quencher Q are n are attached to the probe extension cycle Taq DNA from the quencher the attached to the 5 Sd 3 reporter dye emission is polymerase cleaves the reporter dye emits its ends of a TagMan probe quenched reporter dye from the characteristic probe fluorescence TaqMan Probes Figure 2 1 How the TaqMan reagents work Applied Biosystems offers two types of TaqMan probes e TaqMan MGB minor groove binder probes with non fluorescent quencher NFQ TaqMan probes with TAMRA dye as quencher IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye asa reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 2 3 Chapter 2 Reagent Overview Applied Biosystems offers the following TaqMan probes for use on the StepOne system or the StepOnePlus system Probe Assays 5 Label Dye 3 Label Dye MGB System TagMan MGB TagMan Gene FAM dye NFQ Yes StepOne and probe Expression Assays StepOne Plus m systems TagMan SNP FAM and Genotyping Assays VIC dyes TagMan MGB Custom TaqMan Gene FAM dye pr
59. OnePlus Real Time PCR Systems Reagent Guide Glossary 9 Glossary relative standard curve method Remote Monitor replicate group replicates reporter reverse primer reverse transcriptase Rn ROX dye rs number run method sample Sample DNA 10X Sample Library Glossary 10 Method for determining relative target quantity in samples With the relative standard curve method the StepOne software measures amplification of the target and of the endogenous control in samples in a reference sample and in a standard dilution series Measurements are normalized using the endogenous control Data from the standard dilution series are used to generate the standard curve Using the standard curve the software interpolates target quantity in the samples and in the reference sample The software determines the relative quantity of target in each sample by comparing target quantity in each sample to target quantity in the reference sample A feature in the StepOne software that allows you to monitor a StepOne or StepOnePlus instrument over the network With the Remote Monitor you can monitor the instrument status send an experiment to the instrument monitor amplification plots and temperature plots in real time and download the results to your computer You cannot operate the StepOne or StepOnePlus instrument using the Remote Monitor A set of identical reactions in an experiment Total number of identical reac
60. PCR any target that does not change expression levels with experimental conditions or across samples may serve as an endogenous control Therefore the more targets you have in a singleplex format the higher the probability that you will have one or more suitable endogenous controls against which to normalize your remaining targets Consider the following when choosing between multiplex and singleplex PCR PCR Description Advantage Limitation Singleplex A reaction in which a single target or endogenous control is amplified in the reaction tube or well No optimization is required for TaqMan assays Any target that does not change expression levels with experimental conditions or across samples may serve as an endogenous control Flexibility to use TaqMan or SYBR Green reagents Requires sample for both the target and the endogenous control Multiplex A reaction in which more than one target or endogenous control is amplified in the reaction tube or well Reduces both the running costs and the dependence on accurate pipetting when splitting a sample into two separate tubes The endogenous control assay must be run as a primer limited assay Requires validation and optimization May not be as effective as singleplex PCR when analyzing multiple numbers of targets You cannot use SYBR Green reagents Applied Biosystems StepOne and StepOnePlus Real Time PCR S
61. PCR reactions using the TaqMan SNP Genotyping Assays refer to the TagMan SNP Genotyping Assays Protocol TaqMan Drug Metabolism Genotyping Assays Product TaqMan Drug Metabolism Genotyping Assays are a comprehensive collection of Description Inventoried primer and probe sets for genotyping SNPs insertions and deletions indels and multiple nucleotide polymorphisms MNPs in drug metabolism related genes The assays Use TaqMan reagents to amplify and detect specific polymorphisms in purified genomic DNA samples Are designed using Applied Biosystems bioinformatics pipeline and software as well as genomic information from public SNP databases and public genome assemblies Are designed and optimized to work with Applied Biosystems TaqMan master mixes Product All TaqMan Drug Metabolism Genotyping Assays require Requirements Three components 3 to 30 ng of purified genomic DNA sample 20X Drug Metabolism Genotyping Assay Mix specific for each polymorphism Each assay consists of sequence specific forward and reverse primers to amplify the polymorphic sequence of interest and two TaqMan MGB probes One probe labeled with VIC dye detects the Allele 1 sequence one probe labeled with FAM dye detects the Allele 2 sequence TaqMan Genotyping Master Mix or TaqMan 2X Universal PCR Master Mix with or without AmpErase UNG Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent
62. Plus system See threshold cycle Cy Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Glossary 3 Glossary cycling stage data collection delta Rn ARn derivative reporter C Rn Design Wizard diluent Diluted Sample Concentration 10 for Reaction Mix dilution factor dissociation curve EFF endogenous control endpoint read Glossary 4 In the thermal profile a stage that is repeated A cycling stage is also called an amplification stage For cycling stages you can enable AutoDelta settings See also amplification stage A process during the instrument run in which an instrument component detects fluorescence data from each well of the reaction plate The instrument transforms the signal to electronic data and the data are saved in the experiment file In the StepOne software a data collection point is indicated by an icon in the thermal profile Data collection on Data collection off See baseline corrected normalized reporter ARn The negative first derivative of the normalized fluorescence generated by the reporter during PCR amplification In the derivative reporter Rn vs temperature melt curve the derivative reporter signal is displayed in the y axis A feature in the StepOne software that helps you set up your experiment by guiding you through best practices as you enter your experiment design A reagent used to dilute a sample or s
63. Reagents Kit 200 Reactions N808 0228 Note Presence absence experiments are not supported using Fast or SYBR Green master mixes and protocols RNA Quantitation Using 1 Step Kit Part Number RT PCR TaqMan One Step RT PCR Master Mix Reagents Kit 4309169 TaqMan EZ RT PCR Core Reagents N808 0236 Note Use the TagMan EZ RT PCR Core Reagents when a high temperature RT step is required Note Presence absence experiments are not supported using Fast or SYBR Green master mixes and protocols RNA Quantitation Using 2 Step Step Kit Part Number RT PCR PCR step only TagMan Gene Expression Master Mix 4369016 1 Pack 1 x 5 mL 200 reactions TaqMan Gene Expression Master Mix 4369542 10 Pack 10 x 5 mL 2000 reactions TaqMan 2X Universal PCR Master Mix 4304437 RT step only High Capacity cDNA Reverse Transcription 4374966 Kit TagMan Reverse Transcription Reagents N808 234 Both RT and PCR steps TaqMan Gold RT PCR kit N808 232 Note Presence absence experiments are not supported using Fast or SYBR Green master mixes and protocols Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide D 9 Appendix D Reagent Part Numbers D 10 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Bibliography Afonina I Zivarts M Kutyavin I et al 1997 Efficient priming of PCR with short oligonucleotides conjugated to a minor groove binder Nucleic Acids Res
64. SYBR Green reagents e Descriptions and design guidelines for the following experiment types Quantitation experiments Genotyping experiments Presence absence experiments Intended for laboratory staff and principal investigators who perform experiments using the StepOne or StepOnePlus system Guide Purpose and Audience PN Applied Biosystems StepOne and Explains how to perform experiments on the StepOne and 4376786 StepOnePlus Real Time PCR Systems StepOnePlus systems Each Getting Started Guide Getting Started Guide for Genotyping functions as both Experiments e tutorial using example experiment data provided with Applied Biosystems StepOne and the Applied Biosystems StepOne Real Time PCR 4376787 StepOnePlus Real Time PCR Systems Software StepOne software Getting Started Guide for e A guide for your own experiments Presence Absence Experiments Intended for laboratory staff and principal investigators who Applied Biosystems StepOne and eects experiments using the StepOne or StepOnePlus 4376785 StepOnePlus Real Time PCR Systems y Getting Started Guide for Relative Standard Curve and Comparative C Experiments Applied Biosystems StepOne and 4376784 StepOnePlus Real Time PCR Systems Getting Started Guide for Standard Curve Experiments Applied Biosystems StepOne and Explains how to install and maintain the StepOne and 4376782 StepOnePlus Real Time PCR Systems StepOnePlus systems Cen d Netwo
65. agent which blocks amplification of the internal positive control IPC In the StepOne software the task for the IPC target in wells that contain IPC blocking agent See also negative control blocked IPC wells See reference sample See reagents A system layout in which the StepOne or StepOnePlus instrument is directly connected to a colocated computer by the yellow cable In this layout you can control the instrument with the StepOne software on the colocated computer or with the instrument touchscreen Method for determining relative target quantity in samples With the comparative Cy AAC method the StepOne software measures amplification of the target and of the endogenous control in samples and in a reference sample Measurements are normalized using the endogenous control The software determines the relative quantity of target in each sample by comparing normalized target quantity in each sample to normalized target quantity in the reference sample See threshold cycle Cy Dye that 1s not supplied by Applied Biosystems Custom dyes may be adapted for use in experiments on the StepOne and StepOnePlus systems When using custom dyes the custom dye should be added to the Dye Library and a custom dye calibration performed IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOne
66. al PCR Master Mix with or without AmpErase UNG or TaqMan Fast Universal PCR Master Mix No AmpErase UNG Only one PCR amplification step during each PCR cycle and a simultaneous real time reading to obtain results 3 16 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Available Assays Chapter 3 Quantitation Experiments TaqMan Gene Expression Assays are available for human mouse rat Arabidopsis Drosophila C elegans C familiares dog and M mulatta Rhesus genes The part numbers are PN 4331182 for Inventoried assays PN 4351372 for Made to Order assays The prefix of the assay name indicates the species for which the assay was designed Hs for Homo sapiens human Mm for Mus musculus mouse Rn for Rattus norvegicus rat At for Arabidopsis thaliana Dm for Drosophila melanogaster Ce for C elegans Cf for C familiares dog and Rh for M mulatta Rhesus The suffix of the assay name indicates the assay placement as described in the table below Suffix Description m The assay s probe spans an exon junction the assay does not detect genomic DNA _s The assay s primers and probes are designed within a single exon the assay does detect genomic DNA g The assay may detect genomic DNA the assay s primers and probes may be within a single exon _mH The assay was designed to a transcript belonging to a gene family with high sequence homolo
67. ame temperature for each of the VeriFlex blocks The edge of a zone for samples formed by the six independently thermally regulated VeriFlex blocks In the StepOne software the zone boundaries are displayed in the plate layout as thick red lines Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Glossary 15 Glossary Glossary 16 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Index Numerics 1 step RT PCR about 3 8 primers used 3 9 RNA quantitation 3 25 D 4 D 9 2 step RT PCR about 3 8 primers used 3 9 RNA quantitation 3 26 D 4 D 9 A Advanced Setup workflow 1 9 1 10 amplicon sites 3 primer end 3 23 5 probe end 3 22 G C content 3 22 melting temperature 3 22 screening 3 21 selection 3 21 amplicons selecting small 3 22 Assay Design Guidelines about C 1 genotyping experiments C 2 optimize primer concentrations 3 30 optimize probe concentration 3 33 presence absence experiments 5 16 primer and probe 3 21 3 23 quantitation experiments 3 21 C 1 selecting reagents 3 24 thermal cycling conditions 3 27 assay types Custom assays 1 9 1 10 genotyping experiments 4 6 Inventoried Made to Order Assays 1 9 Pre Designed Validated assays 1 10 presence absence experiments 5 6 quantitation experiments 3 12 selecting 1 9 C carryover UNG to minimize 2 7 comparative CT experiments about 3 6 Also see quantitation experiments 3 6 co
68. any eukaryotic species Choice of FAM dye or VIC dye labels primer limited Custom TaqMan Gene Expression Assays Any species or organism Target of your choice Probe is a FAM dye labeled MGB probe Provided in a convenient single 205 tube TaqMan Gene Expression Master Mix Tailored for precise quantitation by real time PCR for routine and challenging experiments Sensitive detection down to 1 copy of target Multiplex PCR for co amplifying two targets in a single reaction Specificity for differentiation between gene family members Validated with TaqMan Gene Expression Assays Simplifies assay implementation by using one reagent for all assays TaqMan 2X Universal PCR Master Mix with or without AmpErase UNG Provides optimal performance for TaqMan assays that use cDNA or DNA as a template Contains components that ensure excellent assay performance Simplifies assay implementation by using one reagent for all assays IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system Note Presence absence experiments are not supported for Fast or SYBR Green master mixes and protocols For guidelines on designing your experiments with Inventoried Made to Order assays see page 5 10 5 7 Chapter 5 Presence Absence Exper
69. at can be used to detect PCR products on the Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems TaqMan reagents below SYBR Green reagents page 2 4 TaqMan Reagents Experiment Types Development of TaqMan Reagents How the TaqMan Reagents Work TaqMan reagents include Applied Biosystems TaqMan assays preformulated mixes that contain probe and primer sets and Applied Biosystems TaqMan master mixes The assays are specific to the target of interest The master mixes contain the remaining components needed for the PCR reaction You can use TaqMan reagents for the following experiment types Quantitation including Standard curve Relative standard curve Comparative Cy AAC Genotyping Presence absence IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used asa reporter or quencher with the StepOnePlus system Initially intercalator dyes were used to measure real time PCR products The primary disadvantage of this detection method is that it detects accumulation of both specific and nonspecific PCR products Real time systems for PCR were improved by the introduction of fluorogenic labeled probes that use the 5 nuclease activity of 180 DNA polymerase The availability of these fluorogenic probes enabled the development of a real time method for detecting only specific amplificati
70. ation Note Note For more documentation see How to Obtain Support on page xii Obtaining The StepOne Software Help describes how to use each feature of the user interface Information from Access the Help from within the software by doing one of the following the Software Help Press F1 e Click in the toolbar Select Help StepOne Software Help To find topics of interest in the Help Review the table of contents Search for a specific topic Search an alphabetized index Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Xi Preface Send Us Your Comments Applied Biosystems welcomes your comments and suggestions for improving its user documents You can e mail your comments to techpubs appliedbiosystems com IMPORTANT The e mail address above is only for submitting comments and suggestions relating to documentation To order documents download PDF files or for help with a technical question go to http www appliedbiosystems com then click the link for Support See How to Obtain Support on page Front Matter xii How to Obtain Support Xii For the latest services and support information for all locations go to http www appliedbiosystems com then click the link for Support At the Support page you can Search through frequently asked questions FAQs Submit a question directly to Technical Support Order Applied Biosystems user documents
71. ative Quantitation of Gene Expression 4303859 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Preface Documents Related to the Reagent Guide Document PN Applied Biosystems High Capacity cDNA Reverse Transcription Kits 4375575 Protocol Custom TaqMan Gene Expression Assays Protocol 4334429 Custom TaqMan Genomic Assays Protocol Submission Guidelines 4367671 Custom TaqMan SNP Genotyping Assays Protocol 4334431 Power SYBR Green PCR Master Mix and RT PCR Protocol 4367218 Pre Developed TaqMan Assay Reagents Allelic Discrimination 4312214 Protocol Primer Express Software Version 3 0 Getting Started Guide 4362460 SYBR Green PCR and RT PCR Reagents Protocol 4304965 SYBR Green PCR Master Mix and RT PCR Reagents Protocol 4310251 TaqMan Drug Metabolism Genotyping Assays Protocol 4362038 TaqMan Exogenous Internal Positive Control Reagents Protocol 4308335 TaqMan Fast Universal PCR Master Mix 2X Protocol 4351891 TaqMan Gene Expression Assays Protocol 4333458 TaqMan Gene Expression Master Mix Protocol 4371135 TaqMan Genotyping Master Mix Protocol 4371131 TaqMan SNP Genotyping Assays Protocol 4332856 TaqMan Universal PCR Master Mix Protocol 4304449 User Bulletin 2 Relative Quantitation of Gene Expression 4303859 Using TaqMan9 Endogenous Control Assays to Select an Endogenous 127AP08 Control for Experimental Studies Applic
72. ch set amplifies a specific target or endogenous control Typically a probe labeled with FAM dye detects the target and a probe labeled with VIC dye detects the endogenous control IMPORTANT SYBR Green reagents cannot be used for multiplex PCR IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system 0 3 Target Primer Set SN d bid Endogenous Control ae Primer Set cDNA Singleplex PCR Multiplex PCR Figure 3 3 Singleplex vs multiplex PCR In order to perform multiplex PCR you must Ensure that the endogenous control you have selected is more abundant lower C value than all of the targets that you are trying to quantify under all conditions Run the endogenous control assay as a primer limited assay The endogenous control assay for the more abundant template in each reaction must be primer limited to avoid competitive PCR that may alter the Cy of the less abundant template Primer Limiting in Multiplex PCR To generate an accurate multiplex assay it is important that the amplification of one species does not dominate amplification of the other species Otherwise the amplification of a highly abundant species can prevent the less abundant species from amplifying efficiently If the less abundant species does not amplify efficiently your exper
73. cleotide polymorphisms AGTTCATCCGGTCA gt MNPs of up to six bases for genotyping AGTTCATATGGTCA studies Annotated as AGTTCAT CC ATIGGTCA 4 14 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 4 Genotyping Experiments The assays Use TaqMan reagents to amplify and detect specific polymorphisms in purified genomic DNA gDNA Are developed using proprietary assay design software Are designed and optimized to work with an Applied Biosystems TaqMan master mix using universal thermal cycling conditions Product All Custom TaqMan SNP Genotyping Assays require Requirements For More Information A submission file that includes your target SNP sequence You create the submission file using free File Builder software then submit the file to the Custom TaqMan Genomic Assays service Three components to 20 ng of purified gDNA sample per well 40X SNP Genotyping Assay or 80X SNP Genotyping Assay specific for each polymorphism Each assay consists of sequence specific forward and reverse primers to amplify the SNP of interest and two TaqMan MGB probes One probe labeled with VIC dye detects the Allele 1 sequence one probe labeled with FAM dye detects the Allele 2 sequence TaqMan Genotyping Master Mix or TaqMan Universal PCR Master Mix with or without AmpErase UNG PCR amplification and an endpoint read to obtain results For inf
74. cript with 1000 to 30 000 fold _gH greater discrimination sensitivity than the closest homologous transcript if they are present at the same copy number in a sample _u The assay s amplicon spans an exon junction and the probe sits completely in one of the spanned exons TaqMan Endogenous Control Assays TaqMan Endogenous Control Assays are available as Inventoried TaqMan Gene Expression Assays PN 4331182 Each assay contains a FAM dye labeled TaqMan MGB probe in a single preformulated 20X tube Individual control assays for all human mouse and rat species various part numbers Each assay contains either a FAM dye labeled TaqMan MGB probe a VIC dye labeled TagMan MGB probe or a TAMRA dye labeled probe TaqMan Endogenous Controls with VIC dye labels are primer limited IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 5 11 Chapter 5 Presence Absence Experiments For More For information on the latest available products and specific product uses refer Information to the Applied Biosystems Web site http www appliedbiosystems com a On the Home page under TaqMan Products select TaqMan Gene Expression Assays b On the Gene Expressio
75. d allowing accurate peak measurement Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 3 33 Chapter 3 Quantitation Experiments a Linear view 1800 1 250 nM Probe 1300 1400 150 nM Probe 1000 54 c 100 nM Probe gt 000 E 50 nM Probe 1000 1 1000 54 000 54 2 0 5 10 15 2 25 Ej 4 b Log view 250 nM Probe 1 000 150 nM Probe 100 nM Probe 50 nM Probe lt 100061 1 000 E2 4 o 1 000 E3 LB Cycle Figure 3 6 Amplification plot linear and log views of probe concentration titration from 50 to 250 nM 3 34 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 3 Quantitation Experiments For More Information For information on Using the TaqMan reagents refer to TaqMan Fast Universal PCR Master Mix 2X Protocol TaqMan Gene Expression Master Mix Protocol TaqMan Universal PCR Master Mix Protocol Using the SYBR Green reagents refer to Power SYBR Green PCR Master Mix and RT PCR Protocol SYBR Green PCR Master Mix and RT PCR Reagents Protocol SYBR Green PCR and RT PCR Reagents Protocol Performing quantitation experiments on the StepOne and StepOnePlus systems refer to Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Getting Started Guide for Standard Curve Experiments Applied Biosystems StepOne and StepOneP
76. dard curve method standard dilution series standard quantity starting quantity Glossary 12 A system layout in which the StepOne or StepOnePlus instrument is not connected to a computer by the yellow cable In this layout you control the instrument only with the instrument touchscreen and you use a USB drive or network connection to transfer data between the instrument and computer Sample that contains known standard quantities Standard reactions are used in quantitation experiments to generate standard curves See also standard curve and standard dilution series In standard curve and relative standard curve experiments The best fit line in a plot of the Cy values from the standard reactions plotted against standard quantities See also regression line A set of standards containing a range of known quantities Results from the standard curve reactions are used to generate the standard curve The standard curve is defined by the number of points in the dilution series the number of standard replicates the starting quantity and the serial factor See also standard dilution series Method for determining absolute target quantity in samples With the standard curve method the StepOne software measures amplification of the target in samples and in a standard dilution series Data from the standard dilution series are used to generate the standard curve Using the standard curve the software interpolates the absol
77. dividually or as part of the SYBR Green PCR Core Reagents kit Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 2 7 Chapter 2 Reagent Overview General PCR Use the following precautions to minimize sample contamination and PCR product Practices carryover Weara clean lab coat not previously worn while handling amplified PCR products or used during sample preparation and clean gloves when preparing samples for PCR amplification Change gloves whenever you suspect that they are contaminated Maintain separate areas dedicated equipment and supplies for Sample preparation PCR setup Never bring amplified PCR products into the PCR setup area PCR amplification Analysis of PCR products Open and close all sample tubes carefully Avoid splashing or spraying PCR samples Use positive displacement or air displacement pipettors with filter plugged tips Change tips after each use Keep reactions and components capped as much as possible Clean lab benches and equipment periodically with 10 bleach solution or 70 ethanol 2 8 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Quantitation Experiments This chapter covers Section 3 1 About Quantitation Experiments 00 0 0 cece ener 3 3 Section 3 2 Design Guidelines yoy a ica xed a eed eas dos 3 15 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reage
78. e Preface Examples of the user attention words appear below Note The Calibrate function is also available in the Control Console IMPORTANT To verify your client connection you need a valid user ID How to Obtain More Information Related Other StepOne and StepOnePlus System Documents Documentation The documents listed in the table below are not shipped with the StepOne or StepOnePlus instrument Document PN Applied Biosystems StepOne and StepOnePlus Real Time PCR 4376791 Systems Installation Performance Verification Protocol Applied Biosystems StepOne and StepOnePlus Real Time PCR 4376790 Systems Installation Qualification Operation Qualification Protocol Applied Biosystems StepOne and StepOnePlus Real Time PCR 4376788 Systems Planned Maintenance Protocol Documents Related to Genotyping Experiments Document PN Allelic Discrimination Pre Developed TaqMan Assay Reagents Quick 4312212 Reference Card Custom TaqMan Genomic Assays Protocol 4367671 Custom TaqMan SNP Genotyping Assays Protocol 4334431 Ordering TaqMan SNP Genotyping Assays Quick Reference Card 4374204 Performing a Custom TaqMan SNP Genotyping Assay for 96 Well 4371394 Plates Quick Reference Card Performing a TaqMan Drug Metabolism Genotyping Assay for 96 Well 4367636 Plates Quick Reference Card Pre Developed TaqMan Assay Reagents Allelic Discrimination 4312214 Protocol
79. e FAM dye labeled TaqMan Endogenous Control Assays are included as Inventoried TaqMan Gene Expression Assays Optimized preformulated ready to use endogenous control assays Cost effective gene expression quantitation for human mouse rat Arabidopsis Drosophila and any eukaryotic species Choice of FAM dye or VIC dye labels primer limited Custom TaqMan Gene Expression Assays Any species or organism Target of your choice Probe is a FAM dye labeled MGB probe Provided in a convenient single 205 tube TaqMan Gene Expression Master Mix Tailored for precise quantitation by real time PCR for routine and challenging experiments Sensitive detection down to 1 copy of target Multiplex PCR for co amplifying two targets in a single reaction Specificity for differentiation between gene family members Validated with TaqMan Gene Expression Assays Simplifies assay implementation by using one reagent for all assays TaqMan 2X Universal PCR Master Mix with or without AmpErase UNG Provides optimal performance for TaqMan assays that use cDNA or DNA as a template Contains components that ensure excellent assay performance The use of one reagent for all assays simplifies the process of assay implementation TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG Provides the same attributes listed above for TaqMan 2 Universal PCR Master Mix
80. e start and end values for the amplification plot You can apply the manual baseline setting to specific wells in the reaction plate An analysis setting in which you enter the threshold value and select whether to use automatic baseline or manual baseline values The software uses the baseline and the threshold values to calculate the threshold cycle C A plot of data collected during the melt curve stage Peaks in the melt curve can indicate the melting temperature Tm of the target or can identify nonspecific PCR amplification In the StepOne software you can view the melt curve as normalized reporter Rn vs temperature or as derivative reporter Rn vs temperature Also called dissociation curve In the thermal profile a stage with a temperature increment to generate a melt curve In melt curve experiments the temperature at which 5096 of the DNA is double stranded and 50 of the DNA 18 dissociated into single stranded DNA The Tm is displayed in the melt curve A plot of the complete spectral contribution of each dye for the selected well s over the duration of the PCR run In the StepOne software the task for targets or SNP assays in wells that contain water or buffer instead of sample No amplification of the target should occur in negative control wells Previously called no template control NTC In presence absence experiments wells that contain IPC blocking agent instead of sample in the PCR reaction No am
81. ead on the StepOne or StepOnePlus instrument consists of three phases 1 Excitation The instrument illuminates all wells of the reaction plate within the instrument exciting the fluorophores in each reaction 1 2 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide About the Filters About the VeriFlex Technology For More Information Chapter 1 Introduction 2 Emission The instrument optics collect the residual fluorescence emitted from the wells of the reaction plate The resulting image collected by the device consists only of light that corresponds to the range of emission wavelengths 3 Collection The instrument assembles a digital representation of the residual fluorescence collected over a fixed time interval The StepOne software stores the raw fluorescent image for analysis After a run the StepOne software uses calibration data spatial dye and background to determine the location and intensity of the fluorescent signals in each read the dye associated with each fluorescent signal and the significance of the signal The StepOne and StepOnePlus systems use the following filters StepOne system StepOnePlus system Filter Dye Filter Dye 1 FAM dye 1 FAM dye SYBR Green dye SYBR Green dye 2 JOE dye 2 JOE dye VIC dye VIC dye 3 ROX dye 3 TAMRA dye NED dye 4 ROX dye The StepOnePlus ins
82. eagents for quantitation experiments Note You cannot perform multiplex PCR using SYBR Green reagents For more information see Select Singleplex or Multiplex PCR on page 3 10 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 2 Reagent Overview Development of Small molecules that bind to double stranded DNA can be divided into two classes SYBR Green those that intercalate DNA and those that bind the minor groove of DNA Higuchi Reagents Higuchi et al 1992 used the intercalator ethidium bromide for real time detection of PCR Hoechst 33258 is an example of a minor groove binding dye whose fluorescence increases when bound to double stranded DNA Higuchi et al 1993 Regardless of the binding method there are at least two requirements for a DNA binding dye for real time detection of PCR products Increased fluorescence when bound to double stranded DNA No inhibition of PCR Applied Biosystems has developed conditions that permit the use of the SYBR Green I dye in PCR without PCR inhibition and with increased sensitivity of detection compared with ethidium bromide How the SYBR The SYBR Green reagents use the SYBR Green I dye to detect PCR products by Green Reagents binding to double stranded DNA formed during the PCR Here is how it works WO Step 1 When added to a sample SYBR Green I dye immediately binds to all double stranded DNA Step 2 During the PCR AmpliTaq G
83. eal Time PCR Systems Reagent Guide Glossary Advanced Setup AIF allele allelic discrimina tion plot amplicon amplification amplification efficiency EFF amplification plot In the StepOne software a feature that allows you to set up your experiment according to your experiment design Advanced Setup provides you with maximum flexibility in the design and setup of your experiment See assay information file AIF For a given target any of the different sequences that occurs in the population Display of data collected during the post PCR read The allelic discrimination plot is a graph of the normalized reporter signal from the allele 1 probe plotted against the normalized reporter signal from the allele 2 probe A segment of DNA amplified during PCR Part of the instrument run in which PCR produces amplification of the target For quantitation experiments fluorescence data collected during amplification are displayed in an amplification plot and the data are used to calculate results For genotyping or presence absence experiments fluorescence data collected during amplification are displayed in an amplification plot and the data can be used for troubleshooting Calculation of efficiency of the PCR amplification The amplification efficiency is calculated using the slope ofthe regression line in the standard curve A slope close to 3 32 indicates optimal 100 PCR amplification efficiency Factors that aff
84. ect amplification efficiency Range of standard quantities To increase the accuracy and precision of the efficiency measurement use a broad range of standard quantities 5 to 6 logs 105 to 10 fold Number of standard replicates To increase the precision of the standard quantities and decrease the effects of pipetting inaccuracies include replicates PCR inhibitors PCR inhibitors in the reaction can reduce amplification and alter measurements of the efficiency Display of data collected during the cycling stage of PCR amplification Can be viewed as Baseline corrected normalized reporter ARn vs cycle Normalized reporter Rn vs cycle Threshold cycle Cr vs well Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Glossary 1 Glossary amplification stage assay Assay ID assay information file AIF assay mix AutoDelta automatic baseline automatic Cy baseline Glossary 2 Part of the instrument run in which PCR produces amplification of the target The amplification stage 15 called a cycling stage in the thermal profile and consists of denaturing primer annealing and polymerization steps that are repeated For quantitation experiments fluorescence data collected during the amplification stage are displayed in an amplification plot and the data are used to calculate results For genotyping or presence absence experiments fluorescence data collected
85. efficiencies for the target assay and the endogenous control assay are approximately equal For More For more information on quantitation methods refer to User Bulletin 2 Relative Information Quantitation of Gene Expression Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 3 7 Chapter 3 Quantitation Experiments Select 1 Step or 2 Step RT PCR About 1 Step RT PCR About 2 Step RT PCR Reverse transcription polymerase chain reaction RT PCR is used to quantify RNA RT PCR can be performed as a 1 step or 2 step procedure In 1 step RT PCR you perform reverse transcription and the PCR in a single buffer system Figure 3 1 The reaction proceeds without the addition of reagents between the RT and PCR steps 1 step RT PCR offers the convenience of a single tube preparation for RT and PCR amplification However the carryover prevention enzyme AmpErase uracil N glycosylase UNG cannot be used with 1 step RT PCR In 1 step RT PCR the presence of UNG would destroy the cDNA as it is being made For information about UNG see Using UNG to Minimize Reamplification of Carryover Products on page 2 7 Primer extended on MRNA BN NNN TNO MRNA 5 cDNA Reverse Primer Synthesis of 1st cDNA strand i 5 CDNA Primer extended on cDNA A Single Forward Tube Cycle Primer 1 Synthesis of 2nd cDNA strand 3 5 PCR amplification of cDNA Forward Primer 5
86. ent Guide D 5 Appendix D Reagent Part Numbers D 6 Master Mixes Master Mix Part Number TaqMan Genotyping Master Mix 1 Pack 1 x 10 mL 400 reactions 4371355 TagMan Genotyping Master Mix 1 Bulk Pack 1 x 50 mL 4371357 2000 reactions TaqMan 2X Universal PCR Master Mix 200 Reactions 4304437 TaqMan 2X Universal PCR Master Mix 2000 Reactions 4326708 10 Pack TaqMan 2X Universal PCR Master Mix 4305719 TagMan 2X Universal PCR Master Mix No AmpErase UNG 200 4324018 Reactions TaqMan 2X Universal PCR Master Mix No AmpErase UNG 2000 4326614 Reactions 10 Pack TaqMan 2X Universal PCR Master Mix No AmpErase UNG 4324020 Note Genotyping experiments are not supported using Fast or SYBR Green master mixes and protocols Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Appendix D Reagent Part Numbers Presence Absence Experiments Inventoried Made to Order Assays Assays TaqMan Exogenous IPC Reagents Product Part Number TaqMan Gene Expression Assays TaqMan Endogenous Control Assays Note FAM dye labeled TagMan Endogenous Control Assays are included as Inventoried TaqMan Gene Expression Assays Custom TaqMan Gene Expression Assays For information on the latest available products and specific product uses refer to the Applied Biosystems Web site http www appliedbiosystems com
87. epOne and StepOnePlus Real Time PCR Systems Reagent Guide 3 19 Chapter 3 Quantitation Experiments For More For information on using the TaqMan reagents refer to Information TaqMan Fast Universal PCR Master Mix 2X Protocol TaqMan Gene Expression Master Mix Protocol TagMan Universal PCR Master Mix Protocol Design the Experiment Use the StepOne Software For More Information 3 20 For Applied Biosystems Inventoried Made to Order assays use the StepOne software to design your quantitation experiments The StepOne software automatically calculates volumes for the Reaction mix components Sample dilutions Standard curve and relative standard curve experiments only Standard dilution series Note To select the Inventoried Made to Order assay type in the StepOne software go to the Reaction Setup screen in either the Design Wizard or Advanced Setup then select Inventoried Made to Order from the Assay Type dropdown menu For information on designing and performing quantitation experiments on the StepOne and StepOnePlus systems refer to Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Getting Started Guide for Standard Curve Experiments Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Getting Started Guide for Relative Standard Curve and Comparative C4 Experiments Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Cha
88. epOnePlus system Assumptions Text Conventions viii User Attention Words This guide assumes that you Are familiar with the Microsoft Windows XP operating system Are familiar with the Internet and Internet browsers Know how to handle DNA and or RNA samples and prepare them for PCR Understand data storage file transfer and copying and pasting Have networking experience if you plan to integrate the StepOne or StepOnePlus system into your existing laboratory data flow This guide uses the following conventions Bold text indicates user action For example Type 0 then press Enter for each of the remaining fields Italic text indicates new or important words and is also used for emphasis For example Before analyzing always prepare fresh matrix A right arrow symbol gt separates successive commands you select from a drop down or shortcut menu For example Select File Open Two user attention words appear in Applied Biosystems user documentation Each word implies a particular level of observation or action as described below Note Provides information that may be of interest or help but is not critical to the use of the product IMPORTANT Provides information that is necessary for proper instrument operation accurate reagent kit use or safe use of a chemical Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guid
89. er Limiting in Multiplex PCR Considering Relative Abundance of the Target and Reference Limiting Primer Matrix To generate an accurate multiplex assay it is important that the amplification of one species does not dominate amplification of the other Otherwise the amplification of a highly abundant species can prevent the less abundant species from amplifying efficiently If the less abundant species does not amplify efficiently your experiment may produce inaccurate results Or in severe cases detection of the less abundant species may be inhibited completely You can avoid this situation by limiting the concentrations of the primers used to amplify the more abundant species thereby turning off the amplification soon after the Cy has been established Primer limitation results in the reaction components common to both assays not being exhausted allowing the amplification of the less abundant species to continue at high efficiency If the more abundant species 1s not known you should determine it before performing multiplex PCR by running both targets in separate tubes Both amplifications should be primer limited if neither species is consistently more abundant In applying the primer limitation to target and endogenous control amplifications the relative abundance of the two species must be considered For quantitation experiments it is possible to use rRNA as an endogenous control The concentration of rRNA in total
90. eractions that may reduce reaction efficiency and produce nonspecific signal in assays using SYBR Green reagents Avoid primer and probe sequences containing runs of four or more G bases Melting Temperature When you select primers and probes with the recommended melting temperature Tm you can use universal thermal cycling conditions Applied Biosystems recommends that the probe Tm be 10 C higher than that of the primers 5 End of Probes Primer Express software does not select probes with a G on the 5 end The quenching effect of a G base in this position will be present even after probe cleavage The presence of a G base can result in reduced fluorescence values ARn that can negatively affect assay performance G bases in positions close to the 5 end but not on it have not been shown to compromise assay performance Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Summary of Primer and MGB Probe Design Guidelines Chapter 3 Quantitation Experiments 3 End of Primers To reduce the possibility of nonspecific product formation ensure that the last five bases on the 3 end of the primers do not contain more than two C and or G bases Under certain circumstances such as a G C rich template sequence you may have to relax this recommendation to keep the amplicon under 150 basepairs in length In general avoid primer 3 ends extremely rich in G and or C bases Probe Guidelines Primer
91. everse Primer TaqMan reagents 900 900 SYBR Green reagents 50 50 A primer optimization matrix allows you to determine that the minimum primer concentration yields the minimum C and maximum AR A primer optimization matrix can help to compensate for nonspecific primer binding which can reduce the amount of primer available to bind at its specific site For quantitation assays using TaqMan reagents you can achieve optimal performance by selecting the primer concentrations that provide the lowest Cy and highest ARn for a fixed quantity of target template Note Although C4 values are the parameter by which quantitative values are assigned in a real time quantitation assays ARn values can also be important when you are trying to obtain maximum sensitivity and reproducibility The results of a typical TaqMan reagent primer optimization matrix experiment are shown in Figure 3 4 on page 3 31 Figure 3 4a shows the amplification plots for all primer concentration combinations in linear view Figure 3 4b shows the same data in log view format The combination of 50 nM forward and reverse primer Plot group C gives both the lowest ARn and highest C4 All other primer combinations that contain a 150 nM concentration of either the forward or reverse primer Plot group B give a reduced ARn All primer combinations that contain at least 300 nM forward and reverse primer Plot group A give both the highest ARn and the lowest
92. f quantitation methods standard curve relative standard curve and comparative Cy AAC4 In quantitation experiments the amount of target in the samples Absolute quantity can refer to copy number mass molarity or viral load Relative quantity refers to the fold difference between normalized quantity of target in the sample and normalized quantity of target in the reference sample A molecule attached to the 3 end of TaqMan probes to prevent the reporter from emitting fluorescence signal while the probe is intact With TaqMan reagents a nonfluorescent quencher minor groove binder NFQ MGB can be used as the quencher With SYBR Green reagents no quencher is used IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system A feature in StepOne and StepOnePlus systems that allows you to run an experiment without entering plate setup information QuickStart requires a colocated layout with the instrument powered on and an intact instrument computer connection Regression coefficient calculated from the regression line in the standard curve The R value indicates the closeness of fit between the standard curve regression line and the individual C4 data points from the standard reactions A value of 1 00 indicates a perfect fit between the regression line and the data points The rate a
93. for each allele The probes contain different fluorescent reporter dyes to differentiate each allele You can use TaqMan minor groove binder MGB probes on the StepOne and StepOnePlus systems Each TaqgMan MGB probe contains A reporter dye at the 5 end of each probe VIC dye is linked to the 5 end of the Allele 1 probe FAM dye is linked to the 5 end of the Allele 2 probe Aminor groove binder MGB This modification increases the melting temperature Tm of probes without increasing probe length Afonina et al 1997 Kutyavin et al 1997 thereby allowing the design of shorter probes Consequently the TaqMan MGB probes exhibit greater differences in Tm values between matched and mismatched probes greater differences in Tm values provide accurate genotyping Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 4 Genotyping Experiments A nonfluorescent quencher NFQ at the 3 end of the probe Because the quencher does not fluoresce real time PCR systems can measure reporter dye contributions more accurately During PCR each probe anneals specifically to its complementary sequence between the forward and reverse primer sites AmpliTaq Gold DNA polymerase can cleave only probes that hybridize to the allele sequence match Cleavage separates the reporter dye from the quencher dye increasing fluorescence of the reporter dye Thus the fluorescence signals generated dur
94. gy The assay provides between 10 C4 and 15 C difference _sH between the target gene and the gene with the closest sequence homology Therefore the assay detects the target transcript with 1000 to 30 000 fold _gH greater discrimination sensitivity than the closest homologous transcript if they are present at the same copy number in a sample _u The assay s amplicon spans an exon junction and the probe sits completely in one of the spanned exons TaqMan Endogenous Control Assays TaqMan Endogenous Control Assays are available as Inventoried TaqMan Gene Expression Assays PN 4331182 Each assay contains a FAM dye labeled TaqMan MGB probe in a single preformulated 20X tube Individual control assays for all human mouse and rat species various part numbers Each assay contains either a FAM dye labeled TaqMan MGB probe a VIC dye labeled TagMan MGB probe or a TAMRA dye labeled probe TaqMan Endogenous Controls with VIC dye labels are primer limited IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 3 17 Chapter 3 Quantitation Experiments For More For information on the latest available products and specific product uses refer Information to the Appl
95. ied Biosystems Web site http www appliedbiosystems com a On the Home page under TaqMan Products select TaqMan Gene Expression Assays b On the Gene Expression Assays amp Arrays page Under Individual Assays select TaqMan Gene Expression Assays This option links to all TaqMan Gene Expression Assays including TaqMan Endogenous Control Assays that contain FAM dye labeled probes or Under Individual Control Assays select TaqMan Endogenous Control Assays This option links to the individual TaqMan Endogenous Control Assays that contain FAM dye labeled TaqMan MGB probes VIC dye labeled TaqMan MGB probes or TAMRA dye labeled probes For information on Custom TaqMan Endogenous Control Assays refer to the Using TaqMan Endogenous Control Assays to Select an Endogenous Control for Experimental Studies Application Note For information on preparing PCR reactions using the TaqMan Gene Expression Assays refer to the TagMan Gene Expression Assays Protocol Custom TaqMan Gene Expression Assays Product Custom TaqMan Gene Expression Assays are TaqMan probe and primer sets that Description are designed synthesized and formulated by the Custom TaqMan Genomic Assays service based on sequence information that you submit Custom TaqMan Gene Expression Assays allow you to perform quantitation experiments on any gene or splice variant in any organism The assays Use TaqMan reagents to amplify and detect the
96. iment may produce inaccurate results or in severe cases detection of the less abundant species may be inhibited completely You can avoid this situation by limiting the concentrations of the primers used to amplify the more abundant species thereby turning off the amplification soon after the Cy has been established However a primer limited assay may be more susceptible to fluctuations in reaction conditions than the primer non limited target assay that it is normalizing For more information see Appendix B Primer Limiting in Multiplex PCR Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Singleplex vs Multiplex PCR Chapter 3 Quantitation Experiments Primer limiting in multiplex PCR becomes increasingly more complex as the number of targets you quantify increases When you analyze multiple numbers of targets it may be more effective to use singleplex PCR for the following reasons In multiplex PCR it may be difficult to find a suitable endogenous control one that Is more abundant than all of the targets you are quantifying Does not change expression levels with experimental conditions or across different samples You would first have to run all of your target assays and endogenous control assays in both the multiplex and singleplex formats then compare Cy values from both formats to determine if there are any effects of the multiplexing on your Cy values In singleplex
97. iments 5 8 Custom Assays Product Attributes Custom TaqMan Probes and Primers Any species or organism Choice of dye labels quenchers and synthesis scales For use with the Primer Express Software and Applied Biosystems Assay Design Guidelines Primer Express Software Software that designs primers and probes for real time PCR TagMan Gene Expression Master Mix Tailored for precise quantitation by real time PCR for routine and challenging experiments Sensitive detection down to 1 copy of target Multiplex PCR for co amplifying two targets in a single reaction Specificity for differentiation between gene family members Validated with TaqMan Gene Expression Assays Simplifies assay implementation by using one reagent for all assays TaqMan 2X Universal PCR Master Mix with or without AmpErase UNG Provides optimal performance for TaqMan assays that use cDNA or DNA as a template Contains components that ensure excellent assay performance Simplifies assay implementation by using one reagent for all assays IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system Note Presence absence experiments are not supported for Fast or SYBR Green master mixes and protocols For guidelines on designing your experiment
98. ing PCR amplification indicate the alleles that are present in the sample Mismatches Between Probe and Allele Sequences Mismatches between a probe and allele Figure 4 1 reduce the efficiency of probe hybridization Furthermore AmpliTaq Gold DNA polymerase is likely to displace the mismatched probe rather than to cleave it to release reporter dye Allele Y 1 egen 90 VIC dye Match Mismatch B FAM dye 9 Quencher Allele X8 2 AmpliTaq Gold DNA Polymerase Match Mismatch GR1556 Figure 4 1 Results from matches and mismatches between allele and probe sequences in genotyping experiments Table 4 1 summarizes the possible results of the genotyping experiment example shown above Table 4 1 Genotyping experiment results A substantial increase in Indicates VIC dye fluorescence only homozygosity for Allele 1 FAM dye fluorescence only homozygosity for Allele 2 both fluorescent signals heterozygosity Workflow Before performing genotyping experiments on the StepOne and StepOnePlus systems prepare for the experiment as follows 1 Select the assay type below 2 Review the design guidelines for the assay type you selected Section 4 2 on page 4 9 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 4 5 Chapter 4 Genotyping Experiments Select the Assay Type Pre Designed Validated Assays 4 6 When you design y
99. ion Cr r threshold cycle for reference amplification Kg Dividing X4 by Ry yields the following expression Xo ONE s Rr R x 1 E Ke Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide A 7 Appendix A Formulas The exact values of X and Ry depend on a number of factors including Reporter dye used in the probe Sequence context effects on the fluorescence properties of the probe Efficiency of probe cleavage Purity of the probe Setting of the fluorescence threshold Therefore the constant K does not have to be equal to 1 Assuming efficiencies of the target and the reference are the same E EQ E X Crx Cr p 96 7 ek Xx 1 Ad K where Xy Xo Ro the normalized quantity of target e AC Cz x Cz p the difference in threshold cycles for target and reference Rearranging gives the following expression Non SE The final step is to divide the Xy for any sample q by the Xy for the reference sample cb Aae EXE P Xxo 1 Bt where e AAC ACz q ACr cb For amplicons designed and optimized according to Applied Biosystems Assay Design Guidelines amplicon size lt 150 bp the efficiency is close to 1 Therefore the quantity of target normalized to an endogenous control and relative to a reference sample is given by 2 AACT Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Prim
100. ion Method About Standard The standard curve method is used to determine the absolute target quantity in Curve samples With the standard curve method the StepOne software measures Experiments amplification of the target in samples and in a standard dilution series Data from the standard dilution series are used to generate the standard curve Using the standard curve the software interpolates the absolute quantity of target in the samples Components The following components are required when setting up PCR reactions for standard curve experiments Sample The sample in which the quantity of the target is unknown Standard A sample that contains known standard quantities used in quantitation experiments to generate standard curves Standard dilution series A set of standards containing a range of known quantities The standard dilution series is prepared by serially diluting standards Replicates The total number of identical reactions containing identical samples components and volumes Negative Controls Wells that contain water or buffer instead of sample template No amplification of the target should occur in negative control wells About Relative The relative standard curve method is used to determine relative target quantity in Standard Curve samples With the relative standard curve method the StepOne software measures Experiments amplification of the target and of the endogenous control in samples
101. itrarily chosen because it has the lowest expression level of the target Relative Standard Curve Results Each c mycy value in the table below is divided by the brain c myc value to give the values in the final column These results indicate the kidney sample contains 5 5x as much c myc mRNA as the brain sample liver 34 2x as much and lung 15 7x as much To determine relative values perform the following steps 1 Average the c myc and GAPDH values from the table below 2 Divide the c myc average by the GAPDH average 3 Designate the reference sample 4 Divide the averaged sample value by the averaged reference sample value Tissue mee ird GAPDH c myCcy c mycy ng Total Raji RNA ng Total Raji RNA Norm to GAPDH Rel to Brain Brain 0 033 0 51 0 043 0 56 0 036 0 59 0 043 0 53 0 039 0 51 0 040 0 52 Average 0 039 0 004 0 54 0 034 0 07 0 008 1 0 0 12 Kidney 0 40 0 96 0 41 1 06 0 41 1 05 0 39 1 07 0 42 1 06 0 43 0 96 Average 0 41 0 016 1 02 0 052 0 40 0 025 5 5 0 35 A 2 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Appendix A Formulas Tissue c myc GAPDH c myCcy c myCy ng Total Raji RNA ng Total Raji RNA Norm to GAPDH Rel to Brain Liver 0 67 0 29 0 66 0 28 0 70 0 28 0 76 0 29 0 70 0 26 0 68 0 27 Average 6 33 23 34 2x2 37 Lung 0 97 0 82 0 92 0 88 0 86 0 78 0 89 0 77 0 94 0 79 0 97 0 80 Average 4 1 9 09 t
102. late that is added to PCR reactions You can use the IPC to distinguish between true negative results that Is the target is absent in the samples and negative results caused by PCR inhibitors incorrect assay setup or reagent or instrument failure TaqMan Gene Expression Assays and TaqMan SNP Genotyping Assays that have been previously manufactured passed quality control specifications and stored in inventory Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Glossary 5 Glossary IPC IPC blocking agent IPC made to order assays manual baseline manual C4 melt curve melt curve stage melting temperature Tm multicomponent plot negative control NC negative control blocked IPC wells negative control IPC wells no amplification control NAC Glossary 6 In presence absence experiments abbreviation for internal positive control IPC In the StepOne software the task for the IPC target in wells that contain the IPC and do not contain IPC blocking agent See also internal positive control IPC Reagent added to PCR reactions to block amplification of the internal positive control IPC See negative control IPC wells TaqMan Gene Expression Assays or TagMan SNP Genotyping Assays that are manufactured at the time of order Only assays that pass manufacturing quality control specifications are shipped An analysis setting in which you enter the baselin
103. lect primers and probes based on the DNA sequence that you provide If you are designing your own assay follow the summary of the primer and probe design guidelines for quantitation experiments on page 3 23 For a detailed discussion of these guidelines see About the Primer and Probe Design Guidelines on page 3 22 IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system Note Even though a probe is not required for SYBR Green I dye detection it is still a good idea to use Primer Express software to select a primer and probe set when you design an assay for SYBR Green reagents Although no probe will be used the primers will meet all required criteria if you need to convert the assay to TaqMan reagents to obtain higher specificity you can find the probe immediately in the original Primer Express software document Selecting an Selecting a good amplicon site ensures amplification of the target mRNA cDNA Amplicon Site for without co amplifying the genomic sequence pseudogenes and other related genes Quantitation SYBR Green reagents can be useful for screening amplicon sites for gene Assays expression Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 3 21 Chapter 3 About Quantitation Experiments the Primer and Probe Design 3 22 Guideli
104. lls contain sample template the presence of the target is not known Negative controls Wells contain water or buffer instead of sample template Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 5 Presence Absence Experiments Setup Well Types No IPC There are two types of wells Mesa Unknown Unknown wells Wells contain sample template the presence of the target is not known Negative control Negative control wells Wells contain water or buffer instead of sample template Endpoint Presence absence experiments are endpoint experiments in which fluorescence data Detection and are collected after the PCR is complete Post PCR Plate Read To aid in troubleshooting presence absence experiments you can use the Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems to perform real time PCR If you use the StepOne and StepOnePlus systems for PCR amplification perform the pre PCR read and post PCR read runs separately How Presence During the PCR the fluorogenic probes anneal specifically to the complementary Absence target between the forward and reverse primer sites on the template DNA Then Experiments during extension AmpliTaq Gold DNA polymerase cleaves the hybridized probes Work in each sample containing the target The cleavage of each matched probe separates the reporter dye from the quencher dye resulting in increased f
105. low 2 Review the design guidelines for the assay type you selected Section 5 2 on page 5 9 Select the Assay Type When you design your experiments with the StepOne software you can select the following assay types for presence absence experiments Inventoried Made to Order page 5 7 Custom page 5 8 This section lists the products available for each assay type Note The assays are specific to the target of interest The master mixes contain the remaining components needed for the PCR reaction 5 6 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Inventoried Made to Order Assays Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 5 Presence Absence Experiments Product Attributes TaqMan Gene Expression Assays Predesigned gene specific primer and probe sets for human mouse rat Arabidopsis Drosophila C elegans C familiares dog and M mulatta Rhesus genes Probe is a FAM dye labeled MGB probe Provided in a convenient single 205 tube Available as Inventoried or Made to Order assays TagMan Endogenous Control Assays Note FAM dye labeled TaqMan Endogenous Control Assays are included as Inventoried TaqMan Gene Expression Assays Optimized preformulated ready to use endogenous control assays Cost effective gene expression quantitation for human mouse rat Arabidopsis Drosophila and
106. lue per the following formula ARn Rn post PCR read Rn pre PCR read where Rn normalized reporter t Kwok and Higuchi 1989 Saiki et al 1985 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 1 7 Chapter 1 Introduction Select the Reagent Type TaqMan Reagents SYBR Green Reagents Other Reagents You can use the following reagent types chemistries on the StepOne and StepOnePlus systems TaqMan reagents SYBR Green reagents Other fluorescent based reagents TaqMan reagents include Applied Biosystems TaqMan assays preformulated mixes that contain probe and primer sets and Applied Biosystems TaqMan master mixes You can use TaqMan reagents for the following experiment types Quantitation including Standard curve Relative standard curve Comparative Cy AAC Genotyping Presence absence IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used asa reporter or quencher with the StepOnePlus system SYBR Green reagents include primers and master mixes that contain SYBR Green dye You can use SYBR Green reagents for quantitation experiments Standard curve Relative standard curve Comparative Cr AAC4 Note You cannot perform multiplex PCR using SYBR Green reagents For more information see Select Singleplex or Multiplex PCR
107. luorescence by the reporter After PCR cycling the StepOne and StepOnePlus instruments read the fluorescence generated during the PCR amplification The fluorescent signals are used to determine the presence or absence of the target in each sample Reporter signals are normalized to the emission of a passive reference as follows Rim Emission Intensity of Target Template Sequence Emission Intensity of Passive Reference Emission Intensity of Internal Positive Control Emission Intensity of Passive Reference Incorporating an IPC An IPC is a second TaqMan probe and primer set added to the reaction plate to detect a low copy constitutive nucleic acid The IPC and the target are amplified simultaneously in the same reaction well If a well does not exhibit amplification the StepOne software uses the positive signal from the IPC to confirm that the well failed to amplify because of a lack of target template rather than because of a pipetting error or inhibition Note Presence absence experiments can be performed without an IPC however the IPC ensures that a failed PCR is not mistaken for a negative test result Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 5 5 Chapter 5 Presence Absence Experiments Workflow Before performing presence absence experiments on the StepOne and StepOnePlus systems prepare for the experiment as follows 1 Select the assay type be
108. lus Real Time PCR Systems Getting Started Guide for Relative Standard Curve and Comparative Cy Experiments Performing presence absence experiments on the StepOne and StepOnePlus systems refer to the Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Getting Started Guide for Presence Absence Experiments Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 3 35 Chapter 3 Quantitation Experiments 3 36 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Genotyping Experiments 4 This chapter covers Section 4 1 About Genotyping Experiments 00 0000 eee er e Section 42 Design Guidelines 2 2 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 4 1 Chapter 4 Genotyping Experiments 4 2 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 4 Genotyping Experiments Section 4 1 About Genotyping Experiments This section covers R o LEERI S ETERA EE cad eek eee EIEEE EE EERO ET ENE 4 4 Selectilie Assay TYPE gt ones simia e ede a OR GER E Re o dc Ros 4 6 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 4 3 Chapter 4 Genotyping Experiments Overview What Isa A genotyping experiment is an endpoint experiment used to determine the genotype Genotyping Experiment Instruments How Genotyping Experiments Work of unkno
109. lymerase Green reagents AmpliTaq Gold DNA Polymerase ensures a robust reaction and it can dramatically reduce the amount of nonspecific product formation A further benefit is the simplification of assay setup which can be performed at room temperature Note The DNA polymerase included in the TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG is capable of very fast hot start PCR The performance is similar to that of the AmpliTaq Gold DNA Polymerase About MultiScribe Reverse Transcriptase is a recombinant Moloney Murine Leukemia MultiScribe Virus MuLV Reverse Transcriptase that reverse transcribes RNA into Reverse complimentary DNA cDNA Transcriptase 3 26 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 3 Quantitation Experiments Use the Recommended Thermal Cycling Conditions Use the thermal cycling conditions recommended for your sample DNA or cDNA page 3 28 RNA using 1 step RT PCR page 3 28 RNA using 2 step RT PCR page 3 29 Note Thermal cycling conditions for Fast reagents differ from thermal cycling conditions for standard reagents About the The StepOnePlus instrument contains six independently thermally regulated VeriFlex VeriFlex blocks to help you optimize your thermal cycling conditions Technology If you are running your experiment on a StepOnePlus instrument you can set a different temperature for one or more of the VeriFlex blocks c
110. m TaqMan SNP Genotyping Assays The Custom TaqMan SNP Genotyping Assays are FAM dye and VIC dye labeled TaqMan MGB probes and primer sets that are designed synthesized and formulated by the Custom TaqMan Genomic Assays service based on sequence information that you submit The assay mix is available in a single preformulated 40X or 80X tube Note To select a SNP assay in the StepOne software go to the SNP Assays screen in the Design Wizard or to the Plate Setup screen in Advanced Setup In the SNP Assays screen or Plate Setup screen you can select an assay from the library or create a new assay User Designed Assays If you want to design your own primers and probes for SNP assays refer to the Primer Express Software Version 3 0 Getting Started Guide IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used asa reporter or quencher with the StepOnePlus system 1 10 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Reagent Overview This chapter covers Overview TaqMan Reagents SYBR Green Reagents Selecting the Appropriate Reagent 1706 000 Minimizing DNA Contaminants 0 20 c eee eee Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 2 Reagent Overview Overview Applied Biosystems has developed two reagent types chemistries th
111. mer concentrations page 3 30 and probe concentrations page 3 33 IMPORTANT These steps provide a rapid and reliable system for assay design and optimization only when used in their entirety Adopt the system as a whole to achieve the highest level of success For a more detailed description of Applied Biosystems Assay Design Guidelines see Appendix C Note To select the Custom assay type in the StepOne software go to the Reaction Setup screen in either the Design Wizard or Advanced Setup then select Custom from the Assay Type dropdown menu Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Formulas This appendix covers Standard Deviation Calculation Using the Standard Curve Method A 2 Standard Deviation Calculation Using the Comparative Method A 5 Formula for Comparative Cy AAC Experiments 0 00 0000 A 7 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide A 1 Appendix A Formulas Standard Deviation Calculation Using the Standard Curve Method Example Comparing Samples with a Reference Sample The normalized amount of target c mycy in the table below is a unitless number that can be used to compare the relative amount of target in different samples One way to make this comparison is to designate one of the samples as a reference sample In the table below brain is designated as the reference sample brain is arb
112. mple target reaction serial factor series slope SNP SNP assay SNP Assay Library spatial calibration stage Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Glossary In the StepOne software a reaction component displayed on the Reaction Mix Calculations tab of the Reaction Setup screen The software assumes the sample or standard is added to the reaction mix at a 10X concentration For example if the reaction volume is 20 uL the calculated volume of sample or standard for 1 reaction is 2 uL In genotyping experiments the combination of which sample to test and which SNP assay to perform in one PCR reaction Each PCR reaction can contain only one sample and one SNP assay In quantitation experiments the combination of which sample to test and which target to detect and quantify in one PCR reaction In the Design Wizard you can detect and quantify only one target in one PCR reaction Use Advanced Setup to detect and quantify more than one target in one PCR reaction In the StepOne software a numerical value that defines the sequence of quantities in the standard curve The serial factor and the starting quantity are used to calculate the standard quantity for each point in the standard curve For example if the standard curve is defined with a serial factor of 1 10 or 10X the difference between any 2 adjacent points in the curve is 10 fold See standard dilution series
113. mponents 3 6 consumables Also see materials required 4 supported 1 4 contamination minimizing DNA 2 7 Custom assay type 1 9 1 10 presence absence experiments 5 16 quantitation experiments 3 21 Custom TaqMan Gene Expression Assays 3 18 5 12 Custom TaqMan SNP Genotyping Assays 4 14 D data about data collection 1 2 Design Wizard workflow 1 9 1 10 designing experiments genotyping 4 14 4 16 presence absence 5 16 quantitation 3 20 3 21 DNA cDNA quantitation thermal cycling conditions 3 28 dye binding methods of 2 5 E endogenous control component of experiment 3 6 endpoint experiments genotyping 4 4 presence absence 5 5 G G C content and amplicon sites 3 22 genotyping experiments assay types 4 6 components 4 4 conclusions for Assay Design Guidelines C 2 described 4 4 designing 4 14 4 16 how they work 4 4 instruments 4 4 mismatches 4 5 selecting master mix 4 13 4 16 TaqMan reagents 2 2 H hairpin loops and primer choice 3 9 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Index 1 Index Inventoried assay type 1 9 IPC 5 4 5 5 M Made to Order assay type 1 9 master mix selecting for genotyping experiments 4 13 4 16 selecting for presence absence experiments 5 15 selecting for quantitation experiments 3 19 melting temperature and amplicon sites 3 22 mismatch in genotyping experiments 4 5 multiplex PCR described 3 10 primer limiting 3 10 rRNA primers B 1
114. ms StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 3 Quantitation Experiments Section 3 2 Design Guidelines This section covers Inventoried Made to Order ASSAYS o cu casi wee does e rhe de namie 3 16 TaqMan Gene Expression Assays e 3 16 Custom TaqMan Gene Expression 4888 00 0c ee eeee 3 18 select the Master MIE e ian daeca ances d REGENS RES ae meee eed 3 19 Design tbe Exnernment ses Pac dea Ee ea OR CI RC UAR NES RU AG oue 3 20 CUBO EBENE Noe cee Uae eke ees 0 add Que gw 3 21 Design Primers and Probes Using Primer Express Software 3 21 Select ihe Respelts uis ce tombe don P em eo p o eade 3 24 Use the Recommended Thermal Cycling Conditions 3 27 Optimize Primer Concenitrall ns sesso oan t RE XE UR Rx RYE 3 30 Optimize the Probe Concentration ed p DER c Ibo aped ng 3 33 For More Woman circo aassar Fre pei WES EAUX WE INTR Gd d 3 35 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 3 15 Chapter 3 Quantitation Experiments Inventoried Made to Order Assays Workflow Ifyou select the Inventoried Made to Order assay type in the StepOne software Applied Biosystems recommends that you follow the workflow below 1 Select the assay e TaqMan Gene Expression Assays below Custom TaqMan Gene Expression Assays page 3 18 2 Select the master mix page 3 19 3 Design the experiment using the
115. n Assays amp Arrays page Under Individual Assays select TaqMan Gene Expression Assays This option links to all TaqMan Gene Expression Assays including TaqMan Endogenous Control Assays that contain FAM dye labeled probes or Under Individual Control Assays select TaqMan Endogenous Control Assays This option links to the individual TaqMan Endogenous Control Assays that contain FAM dye labeled TaqMan MGB probes VIC dye labeled TaqMan MGB probes or TAMRA dye labeled probes For information on Custom TaqMan Endogenous Control Assays refer to the Using TaqMan Endogenous Control Assays to Select an Endogenous Control for Experimental Studies Application Note For information on preparing PCR reactions using the TaqMan Gene Expression Assays refer to the TagMan Gene Expression Assays Protocol Custom TaqMan Gene Expression Assays Product Custom TaqMan Gene Expression Assays are TaqMan probe and primer sets that Description are designed synthesized and formulated by the Custom TaqMan Genomic Assays service based on sequence information that you submit Custom TaqMan Gene Expression Assays allow you to perform presence absence experiments on any gene or splice variant in any organism The assays e Use TaqMan reagents to amplify and detect the target in cDNA samples Are developed using proprietary assay design software e Are designed and optimized to work with an Applied Biosystems TaqMan master mix using u
116. nal from the reporter dye normalized to the fluorescence signal ofthe passive reference An action that you perform before reanalysis to omit one or more wells from analysis Because no algorithms are applied to omitted wells omitted wells contain no results For a set of data 8 datapoint that 15 significantly smaller or larger than the others A dye that produces fluorescence signal Because the passive reference signal should be consistent across all wells it is used to normalize the reporter dye signal to account for non PCR related fluorescence fluctuations caused by minor well to well differences in concentrations or volume Normalization to the passive reference signal allows for high data precision An illustration of the grid of wells and assigned content in the reaction plate In StepOne systems the grid contains 6 rows and 8 columns In StepOnePlus systems the grid contains 8 rows and 12 columns In the StepOne software you can use the plate layout as a selection tool to assign well contents to view well assignments and to view results The plate layout can be printed included in a report exported and saved as a slide for a presentation One standard in a standard curve The standard quantity for each point in the standard curve is calculated based on the starting quantity and serial factor In genotyping experiments a DNA sample with a known genotype homozygous or heterozygous In the StepOne software the ta
117. nce of interest and two TaqMan MGB probes One probe labeled with VIC dye detects the Allele 1 sequence one probe labeled with FAM dye detects the Allele 2 sequence TaqMan Genotyping Master Mix or TaqgMan 2X Universal PCR Master Mix with or without AmpErase UNG Note Allele 1 and 2 control DNA is included with each assay to allow each homozygote signal to be generated on each run For More For information on the latest available products and specific product uses refer Information to the Applied Biosystems Web sites http www allsnps com and or http www appliedbiosystems com a On the Home page under TaqMan Products select TagMan SNP Genotyping Assays 4 12 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 4 Genotyping Experiments b On the SNP Genotyping Assays page under Pre Designed Validated Assays select TaqMan Pre Developed Assay Reagents for Allelic Discrimination TaqMan PDARs for AD For information on preparing PCR reactions using the TaqMan PDARs for Allelic Discrimination refer to the Pre Developed TaqMan Assay Reagents Allelic Discrimination Protocol Select the Master Mix Available Master Applied Biosystems Pre Designed Validated assays for genotyping experiments are Mixes designed to work with the following master mixes TaqMan PDARs for AD contain TagMan 2X Universal PCR Master Mix with AmpErase UNG
118. nes Guidelines The amplicon should span one or more introns to avoid amplification of the target gene in genomic DNA The primer pair should be specific to the target gene to avoid amplification of pseudogenes or other related genes When designing primers use Primer Express software guidelines fno good sequence is found it may be necessary to examine the sequence and redesign the amplicon or simply screen for more sites If the gene you are studying does not have introns it is not possible to design an amplicon that will amplify the mRNA sequence without amplifying the genomic sequence In this case run a control of your RNA sample that has not been reverse transcribed RT minus controls Selection of Small Amplicons An important default parameter in Primer Express software is the selection of amplicons in the 50 to 150 basepair range Small amplicons are favored because they promote high efficiency amplification In addition high efficiency assays enable relative quantitation to be performed using the comparative Cz AAC method Livak and Schmittgen 2001 This method increases sample throughput by eliminating the need for standard curves G C Content Whenever possible select primers and probes in a region with a G C content of 30 to 80 Regions with a G C content gt 80 may not denature well during thermal cycling leading to a less efficient reaction G C rich sequences are susceptible to nonspecific int
119. niversal thermal cycling conditions Product All Custom TaqMan Gene Expression Assays require Requirements A submission file that includes your target sequence You create the submission file using free File Builder software then submit the file to the Custom TaqMan Genomic Assays service Three components 1 to 100 ng of cDNA sample converted from RNA per well with all wells in a study having the same amount of cDNA 20X Gene Expression Assay or 60X Gene Expression Assay specific for each target Each assay consists of two target specific primers and a FAM dye labeled TaqMan MGB probe in a preformulated 20X or 60X mix 1X final concentrations are 250 nM for the probe and 900 nM for each primer 5 12 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 5 Presence Absence Experiments TaqMan Gene Expression Master Mix or TaqMan Universal PCR Master Mix with or without AmpErase UNG For More For information on the latest available products and specific product uses refer Information to the Applied Biosystems Web site http www appliedbiosystems com a On the Home page under TaqMan Products select TaqMan Gene Expression Assays b On the Gene Expression Assays amp Arrays page under Individual Assays select Custom TaqMan Gene Expression Assays Forinformation on ordering Custom TaqMan Gene Expression Assays refer to the Custom TaqMan Genomic
120. nsiderations for selecting 2 6 development 2 5 how they work 2 5 optimizing quantitation experiments 3 31 T TaqMan Drug Metabolism Genotyping Assays 4 11 TaqMan Endogenous Control Assays 3 17 TaqMan Exogenous Internal Positive Control Reagents 5 13 TaqMan Gene Expression Assays 3 16 5 10 TaqMan MGB probes 2 3 usage 2 4 TaqMan PDARs for AD 4 12 TaqMan reagents 1 3 considerations for selecting 2 6 development 2 2 experiment types 2 2 how they work 2 2 TaqMan SNP Genotyping Assays 4 10 thermal cycling conditions 1 step RT PCR 3 28 2 step RT PCR 3 29 DNA cDNA quantitation 3 28 RNA quantitation 3 28 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Index 3 Index Index 4 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide www appliedbiosystems com Worldwide Sales and Support Applied Biosystems vast distribution and service network composed of highly trained support and applications personnel reaches 150 countries on six continents For sales office locations and technical support please call our local office or refer to our Web site at www appliedbiosystems com Headquarters 850 Lincoln Centre Drive Foster City CA 94404 USA Phone 1 650 638 5800 Toll Free In North America 1 800 345 5224 Fax 1 650 638 5884 12 2008 Applied Bibsystems Part Number 4379704 Rev D
121. nt Guide 3 1 Chapter 3 Quantitation Experiments 3 2 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 3 Quantitation Experiments Section 3 1 About Quantitation Experiments This section covers dr o cohen eee dees cet ee ENTA ETEESI 050 6080050 ET 3 4 Selecta Quantitation Method sis isses ere mtem Ret RR RS 3 5 Select 1 5temar 2 Sfen REPOR 23 axuas dip edanxtebreiuaacu eap Ye uda 3 8 Select Simipleplex ar Multiplex POR wis cucawd dhe pet abee 3p thas de up 3 10 Select the Reagent Type jei5064 bei ur ERE ER eb ERE EE Lakes be FE RR 3 12 Belectihe Assay Tepe va wa cick ea oie Saeed S Ce RE ea eRe eee ECA 3 12 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 3 3 Chapter 3 Quantitation Experiments Overview What Is a Quantitation Experiment How Quantitation Experiments Work Workflow A quantitation experiment is a real time experiment that measures the quantity of a target nucleic acid sequence target during each amplification cycle of the polymerase chain reaction PCR The target can be DNA cDNA or RNA Three types of quantitation experiments are discussed in this guide Standard curve page 3 5 Relative standard curve page 3 5 Comparative Cr AAC4 page 3 6 In real time quantitation experiments the reactions are characterized by the point in time during cycling when amplification of a PCR product achieves a fixed
122. obe Expression Assays Custom TaqMan SNP FAM and VIC Genotyping Assays dyes Custom TaqMan Custom assays FAM TET MGB Probe NED or VIC dye Custom TaqMan Custom assays FAM TET or TAMRA No StepOnePlus Probe VIC dye dye system SYBR Green Reagents Experiment Types 2 4 TaqMan MGB Probes Recommended Applied Biosystems recommends the general use of TaqMan MGB probes especially when conventional TaqMan probes exceed 30 nucleotides The TaqMan MGB probes contain A fluorescent reporter dye at the 5 end Generates a signal when cleaved by the 5 nuclease activity of Taq DNA polymerase A nonfluorescent quencher NFQ at the 3 end Allows Real Time PCR Systems to measure the reporter dye contributions more precisely than probes with TAMRA dye because the quencher does not fluoresce A minor groove binder MGB at the 3 end Increases the melting temperature Tm of probes without increasing probe length Afonina et al 1997 Kutyavin et al 1997 thereby allowing the design of shorter probes Consequently the TaqMan MGB probes exhibit greater differences in Tm values between matched and mismatched probes greater differences in Tm values provide accurate genotyping Standard curve Relative standard curve Comparative Cr AAC SYBR Green reagents include primers and master mixes that contain SYBR Green dye You can use SYBR Green r
123. old DNA Polymerase amplifies the target which creates the PCR product or amplicon The double stranded DNA is denatured to single stranded molecules and the SYBR Green I dye is released Step 3 The primers anneal to the single stranded DNA and the Amplitaq Gold DNA Polymerase amplifies the target creating more double stranded DNA As the PCR progresses more amplicon is created Step 4 The SYBR Green I dye then binds to each new copy of double stranded DNA that is generated during each PCR cycle Since the SYBR Green I dye binds to all double stranded DNA the result is an increase in fluorescence intensity proportional to the quantity of double stranded PCR product produced Figures 2 2 below illustrates this process C4 9 9 9 9 Step 1 Reaction setup i i The SYBR Green dye fluoresces when bound to double stranded DNA 9 e Step 2 Denaturation e When the DNA is denatured into single stranded DNA the SYBR Green dye is released and the fluorescence is drastically reduced Step 3 Polymerization E S d During extension primers e e anneal and PCR product m is generated REVERSE PRIMER 3 9 Step 4 Polymerization completed SYBR Green I dye binds to the double stranded product resulting in a net increase in fluorescence detected by the instrument Figure 2 2 How the SYBR Green reagents work Applied Biosystems StepOne and StepOnePlus Real
124. on products TaqMan reagents use a fluorogenic probe to detect a specific PCR product as it accumulates during the PCR Here is how it works Step 1 An oligonucleotide probe is constructed with a fluorescent reporter dye bound to the 5 end and a quencher on the 3 end Step 2 While the probe is intact the proximity of the quencher greatly reduces the fluorescence emitted by the reporter dye by fluorescence resonance energy transfer FRET F rster resonance F rster V T 1948 through space Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 2 Reagent Overview Step 3 If the target is present the probe anneals between primer sites and is cleaved by the 5 nuclease activity of Taq DNA polymerase during extension Cleavage of the probe Separates the reporter dye from the quencher increasing the reporter dye signal Removes the probe from the target strand allowing primer extension to continue to the end of the template strand Thus inclusion of the probe does not inhibit the overall PCR process Step 4 More reporter dye molecules are cleaved from their respective probes with each cycle resulting in an increase in fluorescence intensity proportional to the quantity of amplicon produced The higher the starting copy number of the nucleic acid target the earlier a significant increase in fluorescence is observed Figure 2 1 illustrates this process P
125. or to the Plate Setup screen in Advanced Setup In the SNP Assays screen or Plate Setup screen you can select an assay from the library or create a new assay For More For information on designing and performing genotyping experiments on the Information StepOne and StepOnePlus systems refer to Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Getting Started Guide for Genotyping Experiments Custom Assays Workflow Ifyou are designing a genotyping experiment using Applied Biosystems Custom assays Applied Biosystems recommends that you follow the workflow below 1 Order the assay Custom TagMan SNP Genotyping Assays below 2 Select the master mix page 4 16 3 Design the experiment using the StepOne software page 4 16 Custom TaqMan SNP Genotyping Assays Product Custom TaqMan SNP Genotyping Assays are TaqMan probe and primer sets that Description are designed synthesized and formulated by the Custom TaqMan Genomic Assays service based on sequence information that you submit Custom TaqMan SNP Genotyping Assays allow you to Action Example Perform genotyping studies with any AGTTCATCCATGGTCA gt possible single nucleotide polymorphism AGTTCATACATGGTCA SNP in any organism Annotated as AGTTCAT C AICATGGTCA Detect insertions deletions in dels of upto AGTTCATCCATGGTCA gt six bases for genotyping studies AGTTCATGGTCA Annotated as AGTTCAT CCAT JGGTCA Detect multiple nu
126. ormation on the latest available products and specific product uses refer to the Applied Biosystems Web sites http www allsnps com and or http www appliedbiosystems com a On the Home page under TaqMan Products select TaqMan SNP Genotyping Assays b On the SNP Genotyping Assays page under Custom Assays select Custom TaqMan SNP Genotyping Assays For information on ordering Custom TaqMan SNP Genotyping Assays refer to the Custom TaqMan Genomic Assays Protocol Submission Guidelines For information on preparing PCR reactions using the Custom TaqMan SNP Genotyping Assays refer to the Custom TaqMan SNP Genotyping Assays Protocol Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 4 15 Chapter 4 Genotyping Experiments Select the Master Mix Available Master Mixes For More Information Applied Biosystems Made to Order assays for genotyping experiments are designed to work with the following master mixes Master Mix Part Number TaqMan Genotyping Master Mix 1 Pack 1 x 10 mL 400 reactions 4371355 TagMan Genotyping Master Mix 1 Bulk Pack 1 x 50 mL 4371357 2000 reactions TaqMan 2X Universal PCR Master Mix 200 Reactions 4304437 TagMan 2X Universal PCR Master Mix 2000 Reactions 4326708 10 Pack TagMan 2X Universal PCR Master Mix 4305719 TaqMan 2X Universal PCR Master Mix No AmpErase UNG 200 4324018 Reactions TaqMan 2X Universal P
127. ost PCR stability for high throughput setup and analysis Validated with TaqMan SNP Genotyping Assays Simplifies assay implementation by using one reagent for all assays TaqMan 2X Universal PCR Master Mix with or without AmpErase UNG Provides optimal performance for TaqMan assays that use cDNA or DNA as a template Contains components that ensure excellent assay performance Simplifies assay implementation by using one reagent for all assays Note Genotyping experiments are not supported for Fast or SYBR Green master mixes and protocols For guidelines on designing your experiments with Custom assays see page 4 14 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 4 7 Chapter 4 Genotyping Experiments 4 8 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 4 Genotyping Experiments Section 4 2 Design Guidelines This section covers Pre Designed Validated ASSAyR co cssuo alienae kh SER RR UR dane s 4 10 TagMan SNP Genotyping Assays iie 4 10 TaqMan Drug Metabolism Genotyping Assays sess 4 11 Pre Developed TaqMan Assay Reagents for Allelic Discrimination 4 12 selectis Nos ot MIR osados ace OR aces ORELL e ioi Aoi Cv Ro deg ded 4 13 Design the 2 24 idka bi SRL EE 4 14 4 14 Custom TaqMan SNP Genotyping Assays sse 4 14 select ie Mastelr MIS 2 ados EO
128. osystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 5 Presence Absence Experiments 5 2 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 5 Presence Absence Experiments Section 5 1 About Presence Absence Experiments This section covers Overview Select the Assay Type Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 5 3 Chapter 5 Presence Absence Experiments Overview What Is a Presence Absence Experiment 5 4 A presence absence experiment is an endpoint experiment that indicates the presence or absence of a specific nucleic acid sequence target in a sample The actual quantity of target is not determined Presence absence experiments are commonly used to detect the presence or absence of a pathogen such as a viral or bacterial pathogen For example a presence absence experiment might be used to determine if Salmonella bacteria are present in hamburger meat The results will show if Salmonella bacteria are present or are not present the quantity of bacteria 1s not determined Components PCR reactions for presence absence experiments include the following components Sample The sample in which the presence 01 8 target is unknown Replicates Identical reactions containing identical components and volumes Internal positive control IPC A short synthetic DNA template that is added
129. our experiments with the StepOne software you can select the following assay types for genotyping experiments Pre Designed Validated below Custom page 4 7 This section lists the products available for each assay type Note The assays are specific to the target of interest The master mixes contain the remaining components needed for the PCR reaction Product Attributes TagMan SNP Predesigned assays for high density genome wide Genotyping Assays marker coverage For screening association candidate region candidate gene or fine mapping studies Convenient single tube format TaqMan Drug Metabolism Genotyping Assays e Detect polymorphisms in 220 genes that code for various drug metabolism enzymes and drug transporters For studying single nucleotide polymorphisms SNPs insertions deletions in dels and multinucleotide polymorphisms MNPs Pre Developed TaqMan Assay Reagents for Allelic Discrimination e Genotype purified DNA samples for specific mutations most assays discriminate between two alleles of single nucleotide polymorphisms SNPs e Minor groove binder MGB added for better genotyping e Allele 1 and 2 control DNA included to allow each homozygote signal to be generated on each run e Closed tube system requires no post PCR manipulation or gels TagMan Genotyping Master Mix Optimized for endpoint fluorescence detection in SNP genotyping applications
130. pErase UNG is added to the reactions For TaqMan Gene Expression Master Mix TaqMan 2X Universal PCR Master Mix with AmpErase UNG and Power SYBR Green PCR Master Mix follow these conditions Step Times and Temperatures Initial Steps PCR 40 Cycles AmpliTaq AmpErase 9 2 PCR Step UNG be DNA Melt eben Activation o ymerase xen Activation HOLD HOLD CYCLE 2 min 6 50 C 10 min 6 95 0 15sec 6 95 0 1 min 6 0 3 29 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 3 Quantitation Experiments Optimize Primer Concentrations Default Primer Concentrations 3 30 Primer Optimization Matrix TaqMan Reagents By independently varying forward and reverse primer concentrations you can identify the concentrations that provide optimal assay performance Primers are always in large molar excess during the exponential phase of PCR amplification When you use TaqMan Gene Expression Master Mix or TaqMan 2X Universal PCR Master Mix Applied Biosystems recommends the primer concentrations listed in Default Primer Concentrations below Detailed discussions follow for the Primer optimization matrix below TagMan reagents below SYBR Green reagents page 3 31 The recommended starting primer concentrations listed in the table below are for DNA and cDNA quantitation assays Concentrations nM Reagent Forward Primer R
131. parisons against known standard quantities Because a standard curve must be constructed for each target standard curve experiments require more reagents and more space in the reaction plate Relative standard curve Uses a standard curve to determine the change in expression of a target in a sample relative to the same target in a reference sample Best for assays that have suboptimal PCR efficiency Requires the least amount of validation because the PCR efficiencies of the target and endogenous control do not need to be equivalent Because a standard curve must be constructed for each target relative standard curve experiments require more reagents and more space in the reaction plate Comparative Cy AAC Uses arithmetic formulas to determine the change in expression of a target in a sample relative to the same target in a reference sample Best for high throughput measurements of relative gene expression of many genes in many samples Relative levels of target in samples can be determined without the use of a standard curve provided that the PCR efficiencies of the target and endogenous control are relatively equivalent Reduced reagent usage More space is available in the reaction plate e Suboptimal low PCR efficiency assays may produce inaccurate results Before you use the comparative C method Applied Biosystems recommends that you determine that the PCR
132. plification should occur in negative control blocked IPC wells because the reaction contains no sample and amplification of the IPC is blocked Previously called no amplification control NAC In presence absence experiments wells that contain IPC template and buffer or water instead of sample Only the IPC template should amplify in negative control IPC wells because the reaction contains no sample Previously called IPC See negative control blocked IPC wells Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide no template control NTC nonfluorescent quencher minor groove binder NFQ MGB normalized quantity normalized reporter Rn omit well outlier passive reference plate layout point positive control post PCR read Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Glossary See negative control NC Molecules that are attached to the 3 end of TaqMan probes When the probe is intact the nonfluorescent quencher NFQ prevents the reporter dye from emitting fluorescence signal Because the NFQ does not fluoresce it produces lower background signals resulting in improved precision in quantitation The minor groove binder MGB increases the melting temperature Tm without increasing probe length It also allows the design of shorter probes Quantity of target divided by the quantity of endogenous control Fluorescence sig
133. pt zone zone boundary Glossary In the StepOne software the task for the target or SNP assay in wells that contain the sample you are testing In quantitation experiments the task for the target in wells that contain a sample with unknown target quantities In genotyping experiments the task for the SNP assay in wells that contain a sample with an unknown genotype In presence absence experiments the task for the target in wells that contain a sample in which the presence of the target is not known In presence absence experiments wells that contain a sample and internal positive control IPC The StepOnePlus instrument contains six independently thermally regulated VeriFlex blocks creating up to six different zones for the 96 sample wells After you enable the VeriFlex blocks in the StepOne software you can set a different temperature for one or more of the VeriFlex blocks In the standard curve the value of y where the regression line crosses the y axis The y intercept indicates the expected threshold cycle C4 for a sample with quantity equal to 1 One of up to six sample temperatures among the 96 wells formed by independently thermally regulated VeriFlex blocks during the StepOnePlus instrument run You can set a different temperature for one or more of the VeriFlex blocks or you can set the same temperature for each of the VeriFlex blocks Note For melt curve steps you need to set the s
134. pter 1 Introduction Select the Experiment Type Endpoint vs Real Time Experiments In this guide the term experiment refers to the entire process of performing a run using the StepOne or StepOnePlus system including setup run and analysis You can perform the following types of experiments on the StepOne and StepOnePlus systems Quantitation including Standard curve Relative standard curve Comparative Cy AAC Genotyping Presence absence Note You can also perform melt curve analysis on the StepOne and StepOnePlus systems For more information access the Help from within the StepOne software by clicking in the toolbar or pressing 1 The three experiment types can be categorized as real time or endpoint experiments as described below Category Properties Experiment Type Real time The instrument monitors the progress of the Quantitation PCR as it occurs e Data are collected throughout the PCR process e Reactions are characterized by the point in time during cycling when amplification of a target is first detected Endpoint Data are collected at the end of the PCR Genotyping process e Reactions are characterized by the quantity of the target accumulated at the end of PCR The datapoint is the normalized intensity of the reporter dye or Ry Presence absence Note Some endpoint experiments also include pre PCR datapoints If so the system calculates the delta R AR va
135. pter 3 Quantitation Experiments Custom Assays Workflow If you select the Custom assay type in the StepOne software for a quantitation experiment that is you are designing your own primers and probes Applied Biosystems recommends that you follow the workflow for the Applied Biosystems Assay Design Guidelines 1 Design primers and probes using Primer Express Software below 2 Select the appropriate reagents page 3 24 IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system 3 Use the recommended thermal cycling conditions page 3 27 4 Begin with default primer and probe concentrations If needed optimize the primer concentrations page 3 30 and probe concentrations page 3 33 IMPORTANT These steps provide a rapid and reliable system for assay design and optimization only when used in their entirety Adopt the system as a whole to achieve the highest level of success For a more detailed description of Applied Biosystems Assay Design Guidelines see Appendix C Note To select the Custom assay type in the StepOne software go to the Reaction Setup screen in either the Design Wizard or Advanced Setup then select Custom from the Assay Type dropdown menu Design Primers and Probes Using Primer Express Software The Primer Express Software uses recommended parameters to se
136. r Mix No AmpErase UNG 4324020 TagMan PCR Core Reagents Kit N808 0228 Note If you purchase the TagMan Exogenous Internal Positive Control Reagents with TaqMan 2X Universal PCR Master Mix kit PN 4308320 you do not need to purchase the master mix separately Note Presence absence experiments are not supported using Fast or SYBR Green master mixes and protocols Custom Assays Assays Product Part Number Custom TaqMan Probes and Primers For information on the latest available products and specific product uses refer to the Applied Biosystems Web site http www appliedbiosystems com D 8 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Appendix D Reagent Part Numbers DNA or cDNA Quantitation Kit Part Number TaqMan Gene Expression Master Mix 1 Pack 1 x 5 mL 200 reactions 4369016 TaqMan Gene Expression Master Mix 10 Pack 10 x 5 mL 4369542 2000 reactions TaqMan 2X Universal PCR Master Mix 200 Reactions 4304437 TaqMan 2X Universal PCR Master Mix 2000 Reactions 4326708 10 Pack TaqMan 2X Universal PCR Master Mix 4305719 TaqMan 2X Universal PCR Master Mix No AmpErase UNG 200 4324018 Reactions TaqMan 2X Universal PCR Master Mix No AmpErase UNG 2000 4326614 Reactions 10 Pack TaqMan 2X Universal PCR Master Mix No AmpErase UNG 4324020 TagMan PCR Core
137. r Mix Part Number TaqMan Gene Expression Master Mix 1 Pack 1 x 5 mL 4369016 200 reactions TaqMan Gene Expression Master Mix 10 Pack 10 x 5 mL 4369542 2000 reactions TaqMan 2X Universal PCR Master Mix 200 Reactions 4304437 TaqMan 2X Universal PCR Master Mix 2000 Reactions 4326708 10 Pack TaqMan 2X Universal PCR Master Mix 4305719 TaqMan 2X Universal PCR Master Mix No AmpErase UNG 200 4324018 Reactions TaqMan 2X Universal PCR Master Mix No AmpErase UNG 2000 4326614 Reactions 10 Pack TaqMan 2X Universal PCR Master Mix No AmpErase 4324020 UNG TagMan PCR Core Reagents Kit N808 0228 Note If you purchase the TagMan Exogenous Internal Positive Control Reagents with TaqMan 2X Universal PCR Master Mix kit PN 4308320 you do not need to purchase the master mix separately Note Presence absence experiments are not supported for Fast or SYBR Green master mixes and protocols For More For information on using the TaqMan reagents refer to the Information TaqMan Gene Expression Master Mix Protocol TagMan Universal PCR Master Mix Protocol Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 5 15 Chapter 5 Presence Absence Experiments Design the Experiment Use the StepOne Software For More Information For Applied Biosystems Inventoried Made to Order assays use the StepOne software to design your presence absence experiments The StepOne software
138. reating up to six different zones for samples or you can set the same temperature for each of the VeriFlex blocks For more information on using the VeriFlex sample blocks access the Help from within the StepOne software by clicking in the toolbar or pressing 1 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 3 27 Chapter 3 Quantitation Experiments DNA or cDNA Quantitation RNA Quantitation Using 1 Step RT PCR 3 28 For TaqMan Fast Universal PCR Master Mix 2X No AmpErase UNG follow these conditions Times and Temperatures Initial Steps PCR 40 Cycles AmpErase UNG SN Activationt Activation Melt Anneal Extend HOLD HOLD CYCLE 2 min 6 50 C 20 sec 95 C 1 sec 6 95 C 20 sec 60 C t Required only if AmpErase UNG is added to the reactions For TaqMan Gene Expression Master Mix TaqMan 2X Universal PCR Master Mix and Power SYBR Green PCR Master Mix follow these conditions Times and Temperatures Initial Steps PCR 40 Cycles AmpliTaq Gold no nont DNA Polymerase Melt Anneal Extend Activation HOLD HOLD CYCLE 2 min 6 50 C 10 min 6 95 C 15 sec 6 95 C 1 min 6 60 C Required only if AmpErase UNG is added to the reactions or is included in the master mix For the TaqMan One Step RT PCR Master Mix Reagents Kit and Power SYBR Green RT PCR Reagents Kit follow these conditions Times and
139. ression Assays Made to Order Predesigned FAM dye labeled TaqMan MGB probe and primer sets that are manufactured at the time of order The assay mix is available in a single preformulated 20X tube Custom TaqMan Gene Expression Assays FAM dye labeled TaqMan MGB probe and primer sets that are designed synthesized and formulated by the Custom TagMan Genomic Assays service based on sequence information that you submit The assay mix is available in a single preformulated 20X or 60X tube Note To select the assay type in the StepOne software go to the Reaction Setup screen in either the Design Wizard or Advanced Setup then select Inventoried Made to Order from the Assay Type dropdown menu Custom Assays For quantitation and presence absence experiments select the Custom assay type in the StepOne software 11 you are designing your own assays primers and probes with Primer Express Software and TaqMan or SYBR Green reagents When designing your own assays follow the Applied Biosystems Assay Design Guidelines to optimize results IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used asa reporter or quencher with the StepOnePlus system Note To select the assay type in the StepOne software go to the Reaction Setup screen in either the Design Wizard or Advanced Setup then select Custom from the Assay Type dropdown menu Applied Bios
140. rking and Maintenance Intended for laboratory staff responsible for the installation and maintenance of the StepOne or StepOnePlus system Applied Biosystems StepOne and 4376783 StepOnePlus Real Time PCR Systems Installation Quick Reference Card Applied Biosystems StepOne and Provides information about the reagents you can use on the 4379704 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide vii Preface Guide Purpose and Audience PN Applied Biosystems StepOne and Explains how to prepare your site to receive and install the 4376768 StepOnePlus Real Time PCR Systems StepOne and StepOnePlus systems Site Preparation Guide Intended for personnel who schedule manage and perform the tasks required to prepare your site for installation of the StepOne or StepOnePlus system Applied Biosystems StepOne Real Time Explains how to use the StepOne software to NA PCR Software Help e Setup run and analyze experiments using the StepOne and StepOnePlus systems Monitor networked StepOne and StepOnePlus instruments e Calibrate StepOne and StepOnePlus instruments e Verify the performance of StepOne and StepOnePlus instruments with an RNase P run Intended for Laboratory staff and principal investigators who perform experiments using the StepOne or StepOnePlus system Laboratory staff responsible for the installation and maintenance of the StepOne or St
141. s with Custom assays see page 5 16 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 5 Presence Absence Experiments Section 5 2 Design Guidelines This section covers Inventoried Made to Order Assays isssesal ien suae eR nena de canes 5 10 TaqMan Gene Expression Assays eee 5 10 Custom TaqMan Gene Expression 4888 cece 5 12 TaqMan Exogenous Internal Positive Control Reagents 5 13 Mie Masker MES adi ev OCT Rp RR AOREA AU eye ee Rides 5 15 Design the nas 0 dew eu idka kE uda 5 16 CCUBLOITI ASSAYS Seed ERR EG ARE REC OM AER Eg RR Go Rd d dod ebd odds 5 16 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 5 9 Chapter 5 Presence Absence Experiments Inventoried Made to Order Assays Workflow Ifyou select the Inventoried Made to Order assay type in the StepOne software Applied Biosystems recommends that you follow the workflow below 1 Select the assay e TaqMan Gene Expression Assays below Custom TaqMan Gene Expression Assays page 5 12 2 Use the TaqMan Exogenous Internal Positive Control Reagents page 5 13 Note Presence absence experiments can be performed without an IPC however the IPC ensures that a failed PCR is not mistaken for a negative test result 3 Select the master mix page 5 15 4 Design the experiment using the StepOne software page 5
142. sk for the SNP assay in wells that contain a sample with a known genotype Used in genotyping and presence absence experiments the part of the instrument run that occurs after amplification In genotyping experiments fluorescence data collected during the post PCR read are displayed in the allelic discrimination plot and used to make allele calls In presence absence experiments fluorescence data collected during the post PCR read are displayed in the presence absence plot and used to make detection calls Also called endpoint read Glossary 7 Glossary pre PCR read primer mix primer probe mix pure dye quantitation method quantity quencher QuickStart R value ramp Glossary 8 Used in genotyping and presence absence experiments the part of the instrument run that occurs before amplification The pre PCR read is optional but recommended Fluorescence data collected during the pre PCR read can be used to normalize fluorescence data collected during the post PCR read PCR reaction component that contains the forward primer and reverse primer designed to amplify the target PCR reaction component that contains the primers designed to amplify the target and a TaqMan probe designed to detect amplification of the target See custom dye and system dye In quantitation experiments the method used to determine the quantity of target in the samples In StepOne and StepOnePlus systems there are three types o
143. ssays Appendix D Reagent Part Numbers Assays Product Part Number TaqMan SNP Genotyping Assays For information on the latest available products and specific product uses TaqMan Drug Metabolism Genotyping Assays refer to the Applied Biosystems Web site Pre Developed TaqMan Assay Reagents for EA Allelic Discrimination http www appliedbiosystems com Master Mixes Master Mix Part Number TaqMan Genotyping Master Mix 1 Pack 1 x 10 mL 400 reactions 4371355 TaqMan Genotyping Master Mix 1 Bulk Pack 1 x 50 mL 4371357 2000 reactions TaqMan 2X Universal PCR Master Mix 200 Reactions 4304437 TaqMan 2X Universal PCR Master Mix 2000 Reactions 4326708 10 Pack TaqMan 2X Universal PCR Master Mix 4305719 TaqMan 2x Universal PCR Master Mix No AmpErase UNG 200 4324018 Reactions TaqMan 2X Universal PCR Master Mix No AmpErase UNG 2000 4326614 Reactions 10 Pack TaqMan 2X Universal PCR Master Mix No AmpErase UNG 4324020 Note Genotyping experiments are not supported using Fast or SYBR Green master mixes and protocols Custom Assays Assays Product Part Number Custom TaqMan SNP Genotyping Assays For information on the latest available products and specific product uses refer to the Applied Biosystems Web site http www appliedbiosystems com Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reag
144. st to direct expression of the native form of the enzyme Kwok and of Carryover Higuchi 1989 Products UNG acts on single and double stranded dU containing DNA It acts by hydrolyzing uracil glycosidic bonds at dU containing DNA sites The enzyme causes the release of uracil thereby creating an alkali sensitive apyrimidic site in the DNA The enzyme has no activity on RNA or dT containing DNA Longo et al 1990 TaqMan Assays For TaqMan assays AmpErase UNG treatment can prevent the reamplification of carryover PCR products from previous PCR reactions When dUTP replaces dTTP in PCR amplification AmpErase UNG treatment can remove up to 200 000 copies of amplicon per 50 uL reaction Note AmpErase UNG also abbreviated as UDG for uracil DNA glycosylase is included in some Applied Biosystems TaqMan master mix formulations and can also be purchased individually When purchasing TaqMan master mixes check the product information to see if the master mix contains AmpErase UNG SYBR Green Dye Assays For SYBR Green I dye assays AmpErase UNG treatment can prevent the reamplification of carryover PCR products from previous PCR reactions Although Power SYBR Green PCR Master Mix and SYBR Green PCR Master Mix do not contain AmpErase UNG they contain dUTP and are thus compatible with AmpErase UNG If contamination from PCR carryover is suspected use AmpErase UNG to troubleshoot the problem Note AmpErase UNG can be purchased in
145. t which the temperature changes during the instrument run Except for the melt curve step the ramp is defined as a percentage For the melt curve step the ramp 18 defined as a temperature increment In the graphical view ofthe thermal profile the ramp is indicated by a diagonal line Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide ramp speed raw data plot reaction mix reagents real time PCR reference sample refSNP ID regression coefficients regression line reject well Glossary Speed at which the temperature ramp occurs during the instrument run Available ramp speeds include fast and standard Foroptimal results using the fast ramp speed Applied Biosystems recommends using TaqMan Fast reagents in your PCR reactions Foroptimal results using the standard ramp speed Applied Biosystems recommends using standard reagents in your PCR reactions IMPORTANT TaqMan Fast reagents are not supported for genotyping or presence absence experiments A plot of raw fluorescence signal not normalized for each optical filter A solution that contains all components to run the PCR reaction except for the template sample standard or control The PCR reaction components you are using to amplify the target and to detect amplification Types of reagents used on the StepOne and StepOnePlus systems TaqMan reagents SYBR Green reagents Other reagents Process
146. tandard before adding it to the PCR reaction The diluent can be water or buffer In the StepOne software a field displayed on the Sample Dilution Calculations tab of the Reaction Setup screen For this field enter the sample concentration you want to use to add to the reaction mix for all samples in the experiment 10X for Reaction Mix indicates that the software assumes the sample or standard component of the reaction mix is at a 10X concentration For example if the diluted sample concentration is 50 0 ng uL 10X the final sample concentration in the reaction is 5 ng uL 1X See serial factor See melt curve See amplification efficiency EFF A target or gene that should be expressed at similar levels in all samples you are testing Endogenous controls are used in relative standard curve and comparative Cy AAC4 experiments to normalize fluorescence signals for the target you are quantifying Housekeeping genes can be used as endogenous controls See also housekeeping gene See post PCR read Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide experiment experiment name experiment type forward primer holding stage housekeeping gene internal positive control IPC inventoried assays Glossary Refers to the entire process of performing a run using the StepOne or StepOnePlus systems including setup run and analysis The types of experiments you can perform
147. target in cDNA samples Are developed using proprietary assay design software e Are designed and optimized to work with an Applied Biosystems TaqMan master mix using universal thermal cycling conditions Product All Custom TaqMan Gene Expression Assays require Requirements A submission file that includes your target sequence You create the submission file using free File Builder software then submit the file to the Custom TaqMan Genomic Assays service Three components 1 to 100 ng of cDNA sample converted from RNA per well with all wells in a study having the same amount of cDNA 20X Gene Expression Assay or 60X Gene Expression Assay specific for each target Each assay consists of two target specific primers and a FAM dye labeled TaqMan MGB probe in a preformulated 20X or 60X mix 1X final concentrations are 250 nM for the probe and 900 nM for each primer 3 18 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 3 Quantitation Experiments TaqMan Gene Expression Master Mix TaqMan Universal PCR Master Mix with or without AmpErase UNG or TaqMan Fast Universal PCR Master Mix No AmpErase UNG Only one PCR amplification step during each PCR cycle and a simultaneous real time reading to obtain results For More For information on the latest available products and specific product uses refer Information to the Applied Biosystems Web site http
148. ter Mix with or without AmpErase UNG 5 10 Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Available Assays Chapter 5 Presence Absence Experiments TaqMan Gene Expression Assays are available for human mouse rat Arabidopsis Drosophila C elegans C familiares dog and M mulatta Rhesus genes The part numbers are PN 4331182 for Inventoried assays PN 4351372 for Made to Order assays The prefix of the assay name indicates the species for which the assay was designed Hs for Homo sapiens human Mm for Mus musculus mouse Rn for Rattus norvegicus rat At for Arabidopsis thaliana Dm for Drosophila melanogaster Ce for C elegans Cf for C familiares dog and Rh for M mulatta Rhesus The suffix of the assay name indicates the assay placement as described in the table below Suffix Description m The assay s probe spans an exon junction the assay does not detect genomic DNA _s The assay s primers and probes are designed within a single exon the assay does detect genomic DNA g The assay may detect genomic DNA the assay s primers and probes may be within a single exon _mH The assay was designed to a transcript belonging to a gene family with high sequence homology The assay provides between 10 C4 and 15 C difference _sH between the target gene and the gene with the closest sequence homology Therefore the assay detects the target trans
149. ter 5 Presence Absence Experiments 5 14 Available Kits The following TaqMan Exogenous Internal Positive Control Reagents kits are available from Applied Biosystems Kits Part Number TagMan Exogenous Internal Positive Control Reagents with 4308320 TaqMan 2X Universal PCR Master Mix with VIC dye TaqMan Exogenous Internal Positive Control Reagents 4308323 Note If you are using this kit you will need to purchase one of these TaqMan reagents separately TaqMan 2X Universal PCR Master Mix PN 4304437 TaqMan PCR Core Reagents Kit PN N808 0228 IMPORTANT The kits listed above contain TAMRA dye labeled probes Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system The kits may be used with the StepOnePlus system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 5 Presence Absence Experiments For More For information on preparing PCR reactions using the TaqMan Exogenous Internal Information Positive Control Reagents refer to the ZagMan Exogenous Internal Positive Control Reagents Protocol Select the Master Mix Available Master Applied Biosystems Inventoried Made to Order assays for presence absence Mixes experiments are designed to work with the following master mixes Maste
150. tion method 3 5 selecting master mix 3 19 selecting reagent type 3 12 selecting reagents for Custom assay type 3 24 D 3 D 9 standard curve 3 5 SYBR Green reagents 2 4 3 31 TaqMan Reagents 2 2 R reagents considerations 2 6 other fluorescent based 1 8 selecting 1 8 2 6 selecting for Custom assay type 3 24 D 3 D 9 SYBR Green reagents 1 8 TaqMan reagents 1 8 2 2 real time PCR quantitation experiments 3 4 TaqMan detection process 2 2 reference sample component of experiment 3 5 3 6 relative standard curve experiments about 3 5 Also see quantitation experiments 3 5 components 3 5 replicate component of experiment 3 5 3 6 replicates 4 4 5 4 RNA quantitation Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Index l step RT PCR 3 25 D 4 D 9 U 2 step RT PCR 3 26 D 4 D 9 TP thermal cycling conditions 3 28 3 29 UNG and shares cea LES 2 7 RT PCR Universal Master Mix reagents l step 3 8 polymerase benefit 3 26 2 step 3 8 method comparison 3 7 3 9 S sample 4 4 5 4 samples component of experiment 3 5 3 6 small amplicons selecting 3 22 standard curve experiments about 3 5 Also see quantitation experiments 3 5 components 3 5 standard dilution series component of experiment 3 5 3 6 standards component of experiment 3 5 3 6 StepOne system assay types 1 9 consumables 1 4 data collection 1 2 experiment types 7 filters 1 3 reagenttypes 1 8 SYBR Green reagents 1 3 co
151. tions containing identical components and identical volumes Fluorescent dye used to detect amplification If you are using TaqMan reagents the reporter dye is attached to the 5 end If you are using SYBR Green reagents the reporter dye is SYBR Green dye An oligonucleotide that flanks the 3 end of the amplicon The reverse primer and the forward primer are used together in PCR reactions to amplify the target An enzyme that converts RNA to cDNA Reverse transcriptase is added to the PCR reaction to perform 1 step RT PCR See normalized reporter Rn A dye supplied by Applied Biosystems and precalibrated on the StepOne and StepOnePlus systems ROX dye is used as the passive reference See refSNP ID Definition of the reaction volume and the thermal profile for the StepOne or StepOnePlus instrument run The template that you are testing In the StepOne software a reaction component displayed on the Reaction Mix Calculations tab of the Reaction Setup screen The software assumes the sample DNA is added to the reaction mix at a 10X concentration For example if the reaction volume is 20 uL the calculated volume of sample for 1 reaction is 2 uL In the StepOne software a collection of samples The Sample Library contains the sample name and the sample color Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Sample or Standard 10X sample SNP assay reaction sa
152. to PCR reactions You can use the IPC to distinguish between true negative results and reactions affected by PCR inhibitors incorrect assay setup or a reagent or instrument failure Note Presence absence experiments can be performed without an IPC however the IPC ensures that a failed PCR is not mistaken for a negative test result Negative Controls Wells that contain water or buffer instead of sample template No amplification of the target should occur in negative control wells In the StepOne software you can set up the PCR reactions for the presence absence experiments three different ways Setup Well Types IPC setup There are three types of wells e Unknown IPC wells Wells contain sample template and IPC template the presence of the target is not known Negative control IPC wells Wells contain IPC template and water or buffer instead of sample template in the PCR reaction Only the IPC template should amplify in negative control IPC wells because the reaction contains no sample template Also called PC Negative control blocked IPC wells Wells contain IPC blocking agent instead of sample template in the PCR reaction No amplification should occur in negative control blocked IPC wells because the reaction contains no sample template and amplification of the IPC is blocked Also called no amplification control NAC No IPC There are two types of wells singleplex setup e Unknown wells We
153. trument contains six independently thermally regulated VeriFlex blocks to help you optimize your thermal cycling conditions You can set a different temperature for one or more of the VeriFlex blocks creating up to six different zones for samples or you can set the same temperature for each of the VeriFlex blocks For more information about any of the topics discussed in this guide access the Help from within Applied Biosystems StepOne Real Time PCR Software v2 0 by pressing F1 clicking in the toolbar or selecting Help gt StepOne Software Help Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 1 3 Chapter 1 Introduction Supported Consumables StepOne System The StepOne system supports the consumables listed below These consumables are for use with both standard and Fast reagents protocols IMPORTANT Use only Fast consumables reaction plates tube strips and tubes with the StepOne and StepOnePlus systems even when performing an experiment with standard reagents Consumable Part Number MicroAmp Fast Optical 48 Well Reaction Plate 4375816 e MicroAmp 48 Well Optical Adhesive Film 4375323 and 4375928 MicroAmp Fast 8 Tube Strip 4358293 MicroAmp Optical 8 Cap Strip 4323032 e MicroAmp Fast Reaction Tube with Cap 4358297 MicroAmp Fast 48 Well Tray 4375282 e MicroAmp 48 Well Base Adaptor 4375284 e MicroAmp 96 Well Support Base 4379590
154. ute quantity of target in the samples See also standard and standard curve In standard curve and relative standard curve experiments a set of standards containing a range of known quantities The standard dilution series is prepared by serially diluting standards For example the standard stock is used to prepare the first dilution point the first dilution point 1s used to prepare the second dilution point and so on In the StepOne software the volumes needed to prepare a standard dilution series are calculated by the number of dilution points the number of standard replicates the starting quantity the serial factor and the standard concentration in the stock See also standard curve A known quantity in the PCR reaction n standard curve experiments the quantity of target in the standard In the StepOne software the units for standard quantity can be for mass copy number viral load or other units for measuring the quantity of target In relative standard curve experiments a known quantity in the standard Standard quantity can refer to the quantity of cDNA or the quantity of standard stock in the PCR reaction The units are not relevant for relative standard curve experiments because they cancel out in the calculations When defining a standard curve in the StepOne software corresponds to the highest or lowest quantity Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide step
155. view demonstrating suspected nonspecific amplification in negative control NC wells b Melt curve analysis confirming that product in NC wells has a different melting temperature from the specific product Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 3 Quantitation Experiments Optimize the Probe Concentration Recommended Probe Concentrations For detection by TaqMan probes the recommended probe concentration of 250 nM ensures excellent assay performance However depending on the requirements of the assay a probe optimization experiment can prove useful Note No probe is required for SYBR Green I dye detection The recommended probe concentrations for DNA and cDNA quantitation assays using TaqMan reagents is 250 nM Figure 3 6 shows the results of a probe optimization experiment in which the probe concentration 15 varied from 50 to 250 nM Figure 3 6a shows an increase in ARn as the probe concentration is increased Figure 3 6b shows that the Cz value changes with sufficient probe concentrations To ensure the best reproducibility especially when you want to detect low copy numbers of a target avoid probe limiting concentrations Run the assay at a probe concentration of 250 nM Using a 250 nM concentration you avoid probe limitation and ensure large ARn values Large ARn values indicate a robust assay that is performing at high efficiency giving high product yield an
156. wn samples With this experiment type you can differentiate a single nucleotide polymorphism SNP A genotyping experiment determines if unknown samples are Homozygotes samples having only allele 1 Homozygotes samples having only allele 2 Heterozygotes samples having both allele 1 and allele 2 Components PCR reactions for genotyping experiments include the following components Sample The sample in which the genotype of the target is unknown Optional Replicates Identical reactions containing identical components and volumes Negative Controls Samples that contain water or buffer instead of template also known as no template controls NTCs Negative controls should not amplify Optional Positive controls Samples that contain known genotypes homozygotes for allele 1 homozygotes for allele 2 and heterozygotes for alleles 1 and 2 Genotyping experiments require two steps thermal cycling PCR amplification followed by endpoint detection of the resulting fluorescent signals You can perform the thermal cycling step PCR amplification on the Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems or on a standalone thermal cycler If you use the StepOne and StepOnePlus systems Youcan analyze the PCR which is helpful for troubleshooting Perform the endpoint plate read separately In genotyping experiments the PCR includes a specific fluorescent dye labeled probe
157. www appliedbiosystems com a On the Home page under TaqMan Products select TaqMan Gene Expression Assays b On the Gene Expression Assays amp Arrays page under Individual Assays select Custom TaqMan Gene Expression Assays Forinformation on ordering Custom TaqMan Gene Expression Assays refer to the Custom TagMan Genomic Assays Protocol Submission Guidelines For information on preparing PCR reactions using the Custom TaqMan Gene Expression Assays refer to the Custom TaqMan Gene Expression Assays Protocol Select the Master Mix Available Master Applied Biosystems Inventoried Made to Order assays for quantitation experiments Mixes are designed to work with the following TaqMan master mixes Master Mix Part Number TaqMan Gene Expression Master Mix 1 Pack 1 x 5 mL 4369016 200 reactions TaqMan Gene Expression Master Mix 10 Pack 10 x 5 mL 4369542 2000 reactions TagMan Fast Universal PCR Master Mix 2X No AmpErase UNG 4352042 250 Reactions TaqMan 2X Universal PCR Master Mix 200 Reactions 4304437 TaqMan 2X Universal PCR Master Mix 2000 Reactions 4326708 10 Pack TaqMan 2X Universal PCR Master Mix 4305719 TaqMan 2X Universal PCR Master Mix No AmpErase UNG 200 4324018 Reactions TagMan 2X Universal PCR Master Mix No AmpErase UNG 2000 4326614 Reactions 10 Pack TaqMan 2X Universal PCR Master Mix No AmpErase 4324020 UNG Applied Biosystems St
158. y performance The use of one reagent for all assays simplifies the process of assay implementation TagMan Fast Universal PCR Master Mix 2X No AmpErase UNG Provides the same attributes listed above for TaqMan 2 Universal PCR Master Mix Allows you to run quantitation experiments on the StepOne and StepOnePlus systems in about 35 min Power SYBR Green PCR Master Mix Highly sensitive quantitation enables low copy number detection as few as two copies of a target gene Detects double stranded DNA so specific probes are not required Contains highly purified AmpliTag Gold DNA polymerase LD to minimize nonspecific product formation including primer dimer For use with the Primer Express Software and Applied Biosystems Assay Design Guidelines SYBR Green PCR Master Mix Detects double stranded DNA so specific probes are not required For standard applications when high sensitivity is not required Contains AmpliTaq Gold DNA polymerase to minimize nonspecific product formation including primer dimer For use with the Primer Express Software and Applied Biosystems Assay Design Guidelines IMPORTANT Applied Biosystems does not recommend the use of TAMRA dye as a reporter or quencher with the StepOne system TAMRA dye may be used as a reporter or quencher with the StepOnePlus system For guidelines on designing your experiments with Custom assays see page 3 21 Applied Biosyste
159. ycling conditions for standard reagents 4 Usedefault primer and probe concentrations or optimize primer and probe concentrations When you use Applied Biosystems Assay Design Guidelines you can use default primer and probe concentrations for non multiplex optimized assays or you can optimize primer and probe concentrations IMPORTANT These steps provide a rapid and reliable system for assay design and optimization only when used in their entirety Adopt the system as a whole to achieve the highest level of success Note Applied Biosystems Assay Design Guidelines do not guarantee that all assays will provide the same level of performance and sensitivity Even the most scrupulous design parameters cannot account for all the possible variations between two different assay systems Conclusions for In general the following conclusions can be made when you use the Assay Design Quantitation Guidelines for quantitation experiments Experiments For most TaqMan assays a concentration of 900 nM primers and 250 nM probe results in a highly reproducible and sensitive assay when using DNA or cDNA as a template Due to the nonspecific nature of its detection SYBR Green I dye primer optimization should be bypassed only with caution However if all guidelines are followed concentrations of 50 nM forward and reverse primers generally provide robust amplification with a good level of specificity when using DNA or cDNA as a template Verify
160. ystems Reagent Guide 3 11 Chapter 3 Quantitation Experiments Select the Reagent Type TagMan vs SYBR Green Reagents You can perform quantitation experiments on the StepOne and StepOnePlus systems with TaqMan reagents SYBR Green reagents For information on choosing between TaqMan and SYBR Green reagents see Considerations for Quantitation Experiments on page 2 6 Select the Assay Type When you design your experiments with the StepOne software you can select the following assay types for quantitation experiments Inventoried Made to Order page 3 13 Custom page 3 14 This section lists the products available for each assay type Note The assays are specific to the target of interest The master mixes contain the remaining components needed for the PCR reaction Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Inventoried Made to Order Assays Applied Biosystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide Chapter 3 Quantitation Experiments Product Attributes TaqMan Gene Expression Assays Predesigned gene specific primer and probe sets for human mouse rat Arabidopsis Drosophila C elegans C familiares dog and M mulatta Rhesus genes Probe is a FAM dye labeled MGB probe Provided in a convenient single 20X tube Available as Inventoried or Made to Order assays TagMan Endogenous Control Assays Not
161. ystems StepOne and StepOnePlus Real Time PCR Systems Reagent Guide 1 9 Chapter 1 Introduction Genotyping Pre Designed Validated Assays Experiments For genotyping experiments the Pre Designed Validated assay type includes TaqMan SNP Genotyping Assays Predesigned FAM dye and VIC dye labeled TagMan MGB minor groove binder probes and primer sets that are available as TaqMan Pre Designed SNP Genotyping Assays The TaqMan Pre Designed SNP Genotyping Assays are manufactured at the time of order Made to Order The assay mix is available in a single preformulated 20X tube TaqMan Drug Metabolism Genotyping Assays Predesigned FAM dye and VIC dye labeled TaqMan MGB probes and primer sets that can be purchased off the shelf Inventoried The assay mix is available in a single preformulated 20X tube Pre Developed TaqMan Assay Reagents for Allelic Discrimination TaqMan PDARs for AD Predesigned FAM dye and VIC dye labeled TaqMan MGB probes and primer sets that can be purchased off the shelf Inventoried The assay mix is available in a single preformulated 10X tube Note To select a SNP assay in the StepOne software go to the SNP Assays screen in the Design Wizard or to the Plate Setup screen in Advanced Setup In the SNP Assays screen or Plate Setup screen select an assay from the library or create a new assay Custom Assays For genotyping experiments the Custom assay type includes the Custo

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