EXPRESS One-Step SYBR GreenER Kits
Contents
1. Continued on next page 15 Troubleshooting continued Signals are present in no template controls and or multiple peaks are present in the melting curve graph Template or reagents are contaminated by nucleic acids DNA cDNA Use melting curve analysis and or run the PCR products on a gel after the reaction to identify contaminants See the guidelines for avoiding contamination on page 6 Primer dimers or other primer artifacts are present Use melting curve analysis to identify primer dimers We recommend using validated pre designed primer sets or design primers using dedicated software programs or primer databases Primer contamination or truncated or degraded primers can lead to artifacts Check the purity of your primers by gel electrophoresis 16 Appendix Technical Support On the Web Visit the Invitrogen website at www invitrogen com for e Complete technical support contact information e Technical resources including manuals vector maps and sequences application notes SDSs FAOs formulations citations handbooks etc e Access to the Invitrogen Online Catalog e Additional product information and special offers Contact Us For more information or technical assistance call write fax or email Additional international offices are listed on our website www invitrogen com Corporate Headquarters Japanese Headquarters European Headquarters2. 5791 Van Allen Way LOOP X Bldg 6F Inchinnan Business Park Carlsbad CA 92008 USA 3 9 15 Kaigan 3 Fountain Drive Tel 1 760 603 7200 Minato ku Tokyo 108 0022 Paisley PA4 9RF UK Tel Toll Free 1 800 955 6288 Tel 81 3 5730 6509 Tel 44 0 141 814 6100 Fax 1 760 602 6500 Fax 81 3 5730 6519 Tech Fax 44 0 141 814 6117 E mail E mail E mail tech_support invitrogen com jpinfo invitrogen com eurotech invitrogen com SDS Certificate of Analysis Safety Data Sheets SDSs are available on our website at www invitrogen com sds The Certificate of Analysis provides detailed quality control and product qualification information for each product Certificates of Analysis are available on our website Go to www invitrogen com support and search for the Certificate of Analysis by product lot number which is printed on the box Continued on next page 17 Technical Support continued Limited Warranty 18 Invitrogen a part of Life Technologies Corporation is committed to providing our customers with high quality goods and services Our goal is to ensure that every customer is 100 satisfied with our products and our service If you should have any questions or concerns about an Invitrogen product or service contact our Technical Support Representatives All Invitrogen products are warranted to perform according to specifications stated on the certificate of analysis The Company will replace free of
3. n ml Ea 011 DNase I Amplification Grade 100units e units 18068 015 015 PureLink Micro to Midi Total RNA 50 rxns 12183 018 Purification System TRIzol Reagent 100 ml 15596 026 200 ml oe 018 PureLink 96 Total RNA Purification Kit PureLink 96 Total RNA Purification Kit Total RNA Purification Kit 4 x 96 4x 96 well plates A 96 well plates 12173 011 011 Quant iT RNA Assay Kit Fluorescein NIST Traceable Standard 5x1ml F36915 50 nM Custom Primers visit www invitrogen com oligos 19 Purchaser Notification Limited Use Label License No 5 Invitrogen Technology 20 The purchase of this product conveys to the buyer the non transferable right to use the purchased amount of the product and components of the product in research conducted by the buyer whether the buyer is an academic or for profit entity The buyer cannot sell or otherwise transfer a this product b its components or c materials made using this product or its components to a third party or otherwise use this product or its components or materials made using this product or its components for Commercial Purposes The buyer may transfer information or materials made through the use of this product to a scientific collaborator provided that such transfer is not for any Commercial Purpose and that such collaborator agrees in writing a not to transfer such materials to any third party and b to use such transferred
4. or 6 258 569 or corresponding patent claims outside the United States expressly by implication or by estoppel No right under any other patent claims such as apparatus or system claims and no right to perform commercial services of any kind including without limitation reporting the results of purchaser s activities for a fee or other commercial consideration is hereby granted expressly by implication or by estoppel This product is for research purposes only Diagnostic uses require a separate license from Roche Further information regarding the 5 nuclease licensing program may be obtained from the Director of Licensing Applied Biosystems 850 Lincoln Centre Drive Foster City California 94404 USA 21 References Ausubel F M Brent R Kingston R E Moore D D Seidman J G Smith J A and Struhl K 1994 Current Protocols in Molecular Biology Greene Publishing Associates and Wiley Interscience New York Chou Q Russell M Birch D Raymond J and Bloch W 1992 Prevention of pre PCR mis priming and primer dimerization improves low copy number amplifications Nucl Acids Res 20 1717 1723 Ishiguro T Saitoh J Yawata H Yamagishi H Iwasaki S and Mitoma Y 1995 Homogeneous quantitative assay of hepatitis C virus RNA by polymerase chain reaction in the presence of a fluorescent intercalater Anal Biochem 229 207 Kotewicz M L D Alessio J M Driftmier K M Blodgett K P
5. amount of template may be as low as 0 5 pg RNA should be free of RNase contamination and aseptic conditions should be maintained RNA may be treated with amplification grade DNase I see page 19 to remove any contaminating genomic DNA To isolate total RNA we recommend the PureLink Micro to Midi Total RNA Purification System TRIzol Reagent or the PureLink 96 Total RNA Purification Kit for high throughput applications see page 19 for ordering information When working with RNA e Use disposable individually wrapped sterile plasticware e Use aerosol resistant pipette tips for all procedures e Use only sterile new pipette tips and microcentrifuge tubes e Wear latex gloves while handling reagents and RNA samples to prevent RNase contamination from the surface of the skin e Use proper microbiological aseptic technique when working with RNA e Dedicate a separate set of pipettes buffers and enzymes for RNA work e Use RNase free microcentrifuge tubes If it is necessary to decontaminate untreated tubes soak the tubes overnight in a 0 01 v v aqueous solution of diethylpyrocarbonate DEPC rinse the tubes with sterile distilled water and autoclave the tubes You can use RNase Away Reagent a non toxic solution available from Invitrogen to remove RNase contamination from surfaces For further information on controlling RNase contamination see Ausubel et al 1994 Sambrook et al 1989 Contin
6. charge any product that does not meet those specifications This warranty limits the Company s liability to only the price of the product No warranty is granted for products beyond their listed expiration date No warranty is applicable unless all product components are stored in accordance with instructions The Company reserves the right to select the method s used to analyze a product unless the Company agrees to a specified method in writing prior to acceptance of the order Invitrogen makes every effort to ensure the accuracy of its publications but realizes that the occasional typographical or other error is inevitable Therefore the Company makes no warranty of any kind regarding the contents of any publications or documentation If you discover an error in any of our publications report it to our Technical Support Representatives Life Technologies Corporation shall have no responsibility or liability for any special incidental indirect or consequential loss or damage whatsoever The above limited warranty is sole and exclusive No other warranty is made whether expressed or implied including any warranty of merchantability or fitness for a particular purpose Additional Products Additional Related products are available separately from Invitrogen Products Ordering information is provided below For more information visit our website at www invitrogen com or contact Technical Service page 17 RNase Away Reagent E
7. of 95 C for 3 seconds 60 C for 30 seconds Optional Melting curve analysis 60 C 95 C refer to instrument manual for specific programming Standard Cycling Program 50 C for 5 minutes cDNA synthesis 95 C for 2 minutes 40 cycles of 95 C for 15 seconds 60 C for 1 minute Optional Melting curve analysis 60 C 95 C refer to instrument manual for specific programming 10 Continued on next page Universal Kits Guidelines and Protocols continued One Step Use the protocol below as a general starting point Scale the qRT PCR reaction volume as needed for your real time instrument Universal Mix ROX is recommended for Applied Biosystems instruments and optional for Stratagene instruments see page 8 Bio Rad iCycler instruments use fluorescein instead of ROX for Dynamic Well Factor readings see page 9 1 Setup reactions on ice A standard 20 l reaction size is provided component volumes can be scaled as desired Always prepare a master mix of common components for multiple reactions 20 ul rxn EXPRESS SYBR GreenER qPCR SuperMix Universal 10 ul 10 uM forward primer 200 nM final 0 4 ul 10 uM reverse primer 200 nM final 0 4 ul ROX Reference Dye 25 uM 0 4 pl 0 04 ul EXPRESS SuperScript Mix for One Step SYBR GreenER 0 5 pl Template RNA e g 1 pg 1 ug total RNA 5 ul DEPC treated water to 20 ul Consult instrument documentation The iCycler uses fluorescein instead of R
8. then be referenced for future readings Select the Persistent Well Factor setting when you are entering the cycling program to reference this saved file Dynamic Well Factors For Dynamic Well Factor readings the user must add fluorescein to the qPCR master mix at a final concentration of 10 20 nM Consult your Bio Rad iCycler instruction manual for details Note that if you select the Dynamic Well Factor option the instrument will automatically insert a 90 second incubation at 95 C before the initial 95 C denaturation step Continued on next page Universal Kits Guidelines and Protocols continued Cycling Programs Universal Mix The following one step cycling programs have been developed as a general starting point when using EXPRESS One Step SYBR GreenER qRT PCR Universal Program your real time instrument to perform cDNA synthesis at or above 50 C immediately followed by PCR amplification as shown below The fast cycling program is designed for the AB 7500 in Fast mode Note This mix is highly robust and can be used with a wide range of cycling programs on different instruments If you have an alternative program that you want to use you should test it with this mix Note that your protocol must include an initial 250 C incubation step for UDG inactivation and cDNA synthesis Fast Cycling Program for the AB 7500 in Fast mode 50 C for 5 minutes cDNA synthesis 95 C for 20 seconds 40 cycles
9. CR SuperMixes may be stored at 4 8 C for up to one month EXPRESS One Step SYBR GreenER Universal 11780 200 11780 01K EXPRESS SYBR GreenER qPCR SuperMix 5ml 5x5ml Universal ROX Reference Dye 500 ul 5 x 500 pl EXPRESS SuperScript Mix for One Step SYBR 250 ul 5 x 250 pl GreenER EXPRESS One Step SYBR GreenER with Premixed 11790 200 11790 01K ROX EXPRESS SYBR GreenER qPCR SuperMix with 5 ml 5x5ml Premixed ROX EXPRESS SuperScript Mix for One Step SYBR 250 ul 5 x 250 pl GreenER Product The Certificate of Analysis CofA provides detailed quality Qualification control information for each product The CofA is available on our website at www invitrogen com cofa and is searchable by product lot number which is printed on each box iv Overview Introduction EXPRESS One Step SYBR GreenER Kits provide components for one step reverse transcription and real time quantitative PCR qRT PCR in a convenient format that is compatible with both rapid and standard qPCR cycling conditions The one step format allows cDNA synthesis and PCR in a single tube using gene specific primers and either total RNA or mRNA The RT mix includes SuperScript III Reverse Transcriptase and RNaseOUT Recombinant Ribonuclease Inhibitor in an optimized formulation All EXPRESS SYBR GreenER qPCR SuperMixes include Platinum Tag DNA polymerase SYBR GreenER fluorescent dye MgCb uracil DNA glyco
10. E tech_support invitrogen com For country specific contact information visit our web site at www invitrogen com
11. HT 7300 StepOne StepOnePlus GeneAmp 5700 and PRISM 7000 and 7700 Continued on next page 12 Kits with Premixed ROX Guidelines and Protocols continued Cycling Programs Kits with Premixed ROX The following one step cycling programs have been developed as a general starting point when using EXPRESS One Step SYBR GreenER qRT PCR with Premixed ROX Program your real time instrument to perform cDNA synthesis at or above 50 C immediately followed by PCR amplification as shown below The fast cycling program is designed for the AB 7900HT and StepOne Note This mix is highly robust and can be used with a wide range of cycling programs on different instruments If you have an alternative program that you want to use you should test it with this mix Note that your protocol must include an initial 250 C incubation step for UDG inactivation and cDNA synthesis Fast Cycling Program for AB 7900HT and StepOne 50 C for 5 minutes cDNA synthesis 95 C for 20 seconds 40 cycles of 95 C for 1 second 60 C for 20 seconds Optional Melting curve analysis 60 C 95 C refer to instrument manual for specific programming Standard Cycling Program 50 C for 5 minutes cDNA synthesis 95 C for 2 minutes 40 cycles of 95 C for 15 seconds 60 C for 1 minute Optional Melting curve analysis 60 C 95 C refer to instrument manual for specific programming Continued on next page 13
12. Kits with Premixed ROX Guidelines and Protocols continued One Step qRT PCR Kits with Premixed ROX 14 Use the protocol below as a general starting point for one step qRT PCR Scale the reaction volume as needed for your real time instrument 1 Setup reactions on ice A standard 20 yl reaction size is provided component volumes can be scaled as desired Always prepare a master mix of common components for multiple reactions 20 ul rxn EXPRESS SYBR GreenER qPCR SuperMix with Premixed ROX 10 ul 10 uM forward primer 200 nM final 0 4 pl 10 uM reverse primer 200 nM final 0 4 ul EXPRESS SuperScript Mix for One Step SYBR GreenER 0 5 pl Template RNA e g 1 pg 1 ug total RNA 5 ul DEPC treated water to 20 ul 2 Prepare control reactions as follows No RT controls To test for genomic DNA contamination of the RNA sample do not add the EXPRESS SuperScript Mix No template controls To test for genomic DNA contamination of the enzyme primer mixes do not add template RNA 3 Cap or seal each PCR tube plate and gently mix Make sure that all components are at the bottom of the tube plate centrifuge briefly if needed 4 Place reactions in a real time instrument programmed as described on the previous page Collect data and analyze results 5 Optional The specificity of the PCR products can be checked by agarose gel electrophoresis Troubleshooting Proble
13. OX for Dynamic Well Factor readings 10 20 nM final concentration see page 9 See the table on page 8 for the amount concentration of ROX to use for your specific instrument 2 Prepare control reactions as follows No RT controls To test for genomic DNA contamination of the RNA sample do not add the EXPRESS SuperScript Mix No template controls To test for genomic DNA contamination of the enzyme primer mixes do not add template RNA 3 Caporseal each PCR tube plate and gently mix Make sure that all components are at the bottom of the tube plate centrifuge briefly if needed 4 Place reactions in a real time instrument programmed as described on the previous page Collect data and analyze results 5 Optional The specificity of the PCR products can be checked by agarose gel electrophoresis 11 Kits with Premixed ROX Guidelines and Protocols Introduction This section provides guidelines and protocols for one step qRT PCR using EXPRESS One Step SYBR GreenER with Premixed ROX Additional The following items are supplied by the user Materials e DEPC treated water Required e Gene specific primers see page 5 for design guidelines e Microcentrifuge e Thermal cycler see page 4 for information on compatible thermal cyclers e PCR tubes plates Premixed ROX ROX Reference Dye is included in the SuperMix at a final Concentration concentration of 500 nM which is compatible with Applied Biosystems 7900
14. and Gerard G F 1985 Cloning and overexpression of Moloney murine leukemia virus reverse transcriptase in Escherichia coli Gene 35 249 258 Lindahl T Ljungquist S Siegert W Nyberg B and Sperens B 1977 DNA N glycosidases properties of uracil DNA glycosidase from Escherichia coli J Biol Chem 252 3286 3294 Longo M Berninger M and Hartley J 1990 Use of uracil DNA glycosylase to control carry over contamination in polymerase chain reactions Gere 93 125 128 Sambrook J Fritsch E F and Maniatis T 1989 Molecular Cloning A Laboratory Manual Second Ed Cold Spring Harbor Laboratory Press Plainview New York Sharkey D J Scalice E R Christy K G Atwood S M and Daiss J L 1994 Antibodies as thermolabile switches high temperature triggering for the polymerase chain reaction Biotechnology 12 506 509 Wittwer C T Herrmann M G Moss A A and Rasmussen R P 1997 Continuous fluorescence monitoring of rapid cycle DNA amplification BioTechniques 22 130 138 2010 Life Technologies Corporation All rights reserved For research use only Not intended for any animal or human therapeutic or diagnostic use The trademarks mentioned herein are the property of Life Technologies Corporation or their respective owners 22 invitrogen by Life technologies Corporate Headquarters 5791 Van Allen Way Carlsbad CA 92008 T 1 760 603 7200 F 1 760 602 6500
15. ck polymerase activity at ambient temperatures Chou et al 1992 Sharkey et al 1994 Activity is restored after the initial denaturation step in PCR cycling providing an automatic hot start in qPCR for increased sensitivity specificity and yield UDG and dUTP in the qPCR SuperMix prevent the reamplification of carryover PCR products between reactions Lindahl et al 1977 Longo et al 1990 dUTP ensures that any amplified DNA will contain uracil while UDG removes uracil residues from single or double stranded DNA The UDG used in the kit is a heat labile form of the enzyme that destroys any contaminating dU containing product from previous reactions prior to cDNA synthesis This UDG is inactivated at temperatures of 50 C or higher thereby allowing cDNA synthesis from genuine target sequences when used with a high temperature RT such as SuperScript III Reverse Transcriptase ROX Reference Dye is either premixed in the SuperMix or included as a separate tube in the kit to normalize the fluorescent signal between reactions for instruments that are compatible with this option The following items are supplied by the user e Template RNA e Gene specific primers e DEPC treated water e Microcentrifuge e Thermal cycler e Optional Normalization dye for instruments that do not use ROX e PCR tubes plates Instrument Compatibility Universal Kits Kits with Premixed ROX EXPRESS One Step SYBR GreenER Universal incl
16. esearch use please contact Out Licensing Life Technologies 5791 Van Allen Way Carlsbad California 92008 Phone 760 603 7200 or e mail outlicensing lifetech com Continued on next page Purchaser Notification continued Limited Use Label License No 14 Direct Inhibition by Anti Polymerase Antibodies Limited Use Label License No 274 5 Nuclease Process Licensed to Life Technologies Corporation under U S Patent Nos 5 838 671 5 587 287 and foreign equivalents for use in research only A license to perform the 5 nuclease process for research requires the use of a Licensed 5 Nuclease Kit containing Licensed Probe or the combination of an Authorized Core Kit plus Licensed Probe or license rights that may be purchased from Applied Biosystems This product is an Authorized Core Kit without Licensed Probe Its purchase price includes a limited non transferable immunity from suit under U S Patents and corresponding patent claims outside the United States owned by Roche Molecular Systems Inc or F Hoffmann La Roche Ltd Roche for using only this amount of the product in the practice of the 5 nuclease process solely for the purchaser s own internal research and development activities This product is also an Authorized Core Kit for use with service sublicenses available from Applied Biosystems This product conveys no rights under U S Patents Nos 5 804 375 6 214 979 5 538 848 5 723 591 5 876 930 6 030 787
17. invitrogen by technologies EXPRESS One Step SYBR GreenER Kits For one step qRT PCR using EXPRESS SYBR GreenER qPCR SuperMixes Catalog nos 11780 200 11780 01K 11790 200 and 11790 01K Rev Date 14 July 2010 Manual part no A10326 MAN0000688 ii Table of Contents Kit Contents and Storage sssssssssssssssseeeeeeneee iv OVeEVIOW eH eee I Eee PLE De EIE Een e PE Hee tenis 1 Instrument Compatibility seen 4 Method S scence a aana enana aaan aaan adine aninda inab aran rav R RA ERI RR Ir E Ring CAE haee 5 General qRT PCR Guidelines and Parameters sss 5 Template RNA 4i eio eere ie HE I e RH ra s 6 Universal Kits Guidelines and Protocols esses 8 Kits with Premixed ROX Guidelines and Protocols 12 Troubleshooting eetcaesen endet ede d perde ei dar 15 Appendix qw cescedons isaaeedeceodwennd sevensonatanscenesadnavavenececuecnnaraveananeuedads 17 Technical Support voc Svcd waste ette e e e einer 17 Additional Products ss iiio ote eie on Roi 19 Purchaser Notification eese eene ener 20 Ref rernceS3 uc ci ed en e c e ee estne 22 iii Kit Contents and Storage Kit EXPRESS One Step SYBR GreenER Kits are shipped on Components dry ice The components in each kit are listed below and Storage Storage Store all components at 20 C for long term g P amp storage EXPRESS qP
18. le form of UDG in the SuperMix prevents amplification of carryover PCR products between one step reactions SYBR GreenER fluorescent dye is a double stranded DNA dsDNA binding dye that in this formulation provides higher sensitivity and lower PCR inhibition than SYBR Green I dye It can be used on real time PCR instruments calibrated for SYBR Green I dye without any change of filters or settings In qPCR as dsDNA accumulates SYBR GreenER dye generates a fluorescent signal that is proportional to the DNA concentration Ishiguro et al 1995 Wittwer et al 1997 SuperScript III Reverse Transcriptase is a version of M MLV RT that has been engineered to reduce RNase H activity and provide increased thermal stability for higher yields of cDNA Kotewicz et al 1985 The enzyme in this RT mix formulation can synthesize cDNA at a temperature range of 50 60 C Because SuperScript III RT is not significantly inhibited by ribosomal and transfer RNA it can be used to synthesize cDNA from total RNA RNaseOUT Ribonuclease Inhibitor is included in the SuperScript mix to safeguard against degradation of target RNA due to ribonuclease contamination Continued on next page Overview continued Platinum Taq DNA Polymerase Uracil DNA Glycosylase UDG ROX Reference Dye Additional Materials Required Platinum Taq DNA Polymerase is recombinant Tag DNA polymerase complexed with proprietary antibodies that blo
19. m Cause Solution No PCR productis RNA has been Confirm RNA degradation by evident in the qPCR damaged degraded bioanalyzer or running on a gel and graph or on a gel replace RNA if necessary RNase Maintain aseptic conditions contamination cDNA synthesis SuperScript III in this formulation temperature too typically operates ina temperature high low priming range of 50 60 C efficiency Primers are blocked Raise the incubation temperature by secondary and or redesign primer s structure PCR product is qPCR instrument Confirm that you are using the evident on a gel but settings are correct instrument settings dye not in the qPCR incorrect selection reference dye filters and graph acquisition points Product detected at Inefficient cDNA Adjust cDNA synthesis temperature higher than synthesis and or primer design expected cycle RT inhibitors are Remove inhibitors in the purified number present in RNA RNA by an additional 70 ethanol wash RNA has been Confirm RNA degradation by damaged degraded Bioanalyzer or running on a gel and replace if necessary RNase Maintain aseptic conditions contamination Inefficient PCR Optimize PCR conditions by amplification adjusting annealing temperature and or redesigning the primers Not enough Increase concentration of template template RNA RNA to 10 ng 1 ug total RNA Higher than Too much sample Decrease the concentration of expected signal added to reactions template RNA
20. materials and or information solely for research and not for Commercial Purposes Commercial Purposes means any activity by a party for consideration and may include but is not limited to 1 use of the product or its components in manufacturing 2 use of the product or its components to provide a service information or data 3 use of the product or its components for therapeutic diagnostic or prophylactic purposes or 4 resale of the product or its components whether or not such product or its components are resold for use in research For products that are subject to multiple limited use label licenses the terms of the most restrictive limited use label license shall control Life Technologies Corporation will not assert a claim against the buyer of infringement of patents owned or controlled by Life Technologies Corporation which cover this product based upon the manufacture use or sale of a therapeutic clinical diagnostic vaccine or prophylactic product developed in research by the buyer in which this product or its components was employed provided that neither this product nor any of its components was used in the manufacture of such product If the purchaser is not willing to accept the limitations of this limited use statement Life Technologies is willing to accept return of the product with a full refund For information about purchasing a license to use this product or the technology embedded in it for any use other than for r
21. n of 25 uM ROX AB 7300 7900HT StepOne StepOnePlus 0 4 ul 50X 500 nM and PRISM 7000 and 7700 AB 7500 Stratagene Mx3000P Mx3005P and 0 04 ul 500X 50 nM Mx40009 Continued on next page Universal Kits Guidelines and Protocols continued Fluorescein Bio Rad iCycler instruments require the collection of well for Bio Rad factors before each run to compensate for any instrument iCycler or pipetting non uniformity Well factors for SYBR Instruments GreenER experiments are calculated using an additional fluorophore fluorescein Well factors are collected using either a separate plate containing fluorescein in each well External Well Factors or the experimental plate with fluorescein spiked into the qPCR master mix Dynamic Well Factors You must select the method when you start each run using the iCycler Fluorescein is available separately from Bio Rad or Fluorescein NIST Traceable Standard is available from Invitrogen as a 50 uM solution see page 19 for ordering information External Well Factors The Bio Rad iCycler instruction manual provides instructions on preparing and using the External Well Factor plate The iCycler will automatically insert a 3 cycle program before your experimental cycling program to perform the External Well Factor reading Note The iCycler iQ5 and MyiQ systems allow you to save the data from an External Well Factor reading as a separate file which can
22. sing a bioanalyzer such as the Agilent 2100 bioanalyzer with an RNA LabChip Alternatively total RNA can be analyzed by agarose gel electrophoresis RNA isolated using the PureLink kits or TRIzol Reagent typically has a 28S to 18S band ratio of 21 5 RNA is judged to be intact if discreet 28S and 18S ribosomal RNA bands are observed Universal Kits Guidelines and Protocols Introduction Additional Materials Required ROX Reference Dye Concentration This section provides guidelines and protocols for one step qRT PCR using EXPRESS One Step SYBR GreenER qRT PCR Universal The following items are supplied by the user e DEPC treated water e Genecspecific primers see page 5 for design guidelines e Microcentrifuge e Thermal cycler see page 4 for information on compatible thermal cyclers e PCR tubes plates ROX Reference Dye is supplied as a separate tube in the Universal Kits ROX is recommended for fluorescence normalization on Applied Biosystems instruments and is optional for Stratagene s Mx3000P Mx3005P and Mx4000 It is not required on other instruments ROX is composed of a glycine conjugate of 5 carboxy X rhodamine succinimidyl ester and is supplied at a concentration of 25 uM Use the following table to determine the amount of 25 uM ROX to use with a particular instrument Amount of Effective Fold Final ROX Instrument ROX per 20 1 Concentration Concentration reactio
23. sylase UDG dNTPs with dUTP instead of dTTP and stabilizers Note that this unique one step formulation includes a special heat labile form of UDG in the SuperMix to help prevent reamplification of carryover PCR products between reactions e SuperMix with Premixed ROX The qPCR SuperMix with premixed ROX includes ROX Reference Dye at a final concentration of 500 nM to normalize the fluorescent signal on instruments that are compatible with this option e Universal SuperMix The Universal SuperMix includes ROX as a separate component for instruments that use ROX at a different concentration or do not require ROX Continued on next page Overview continued Advantages of the Kits SYBR GreenER Fluorescent Dye SuperScript Ill Reverse Transcriptase This highly robust one step formulation provides optimal convenience and sensitivity in qRT PCR with sensitive detection and a broad quantification range e SYBR GreenER dye in this formulation provides higher sensitivity and lower PCR inhibition than other fluorescent double stranded DNA binding dyes e SuperScript III Reverse Transcriptase has been engineered for reduced RNase H activity and increased thermal stability for higher yields of CDNA e Platinum Taq DNA Polymerase provides an automatic hot start in PCR for increased sensitivity specificity and yield and has a short activation time for the rapid cycling of fast qPCR instruments e A special heat labi
24. tion at 50 C For problematic templates or to increase the specificity of cDNA priming increase the cDNA synthesis temperature up to 60 C e Forinstrument specific guidelines see the section for each type of SuperMix Gene specific primers are required for one step qRT PCR We strongly recommend using a primer design program such as OligoPerfect available on the Web at www invitrogen com oligos or Vector NTI In addition to designing primers for optimal efficiency programs such as this will automatically perform a BLAST search of NCBI databases to ensure that primers are target specific The amplicon length should be approximately 80 250 bp and the primers should be designed to anneal to exons on both sides of an intron or within the exon exon boundary of the target mRNA to allow differentiation of cDNA from genomic DNA A final concentration of 200 nM per primer is effective for most reactions Optimal results may require a titration of primer concentrations between 100 and 500 nM Melting curve analysis should always be performed following real time qPCR to identify the presence of primer dimers and analyze the specificity of the reaction Program your instrument for melting curve analysis using the instructions provided with your specific instrument Template RNA Input RNA General Handling of RNA Starting material can range from 1 pg to 1 ug of purified total RNA If you are starting with isolated mRNA the
25. udes ROX Reference Dye as a separate tube and can be used with a wide range of real time instruments including the following e Applied Biosystems 7900HT 7300 7500 StepOne StepOnePlus GeneAmp 5700 and PRISM 7000 and 7700 e Bio Rad MJ Research iCycler iQ i105 and MyiQ DNA Engine Opticon and Opticon 2 and Chromo4 Real Time Detector e Cepheid Smart Cycler e Corbett Research Rotor Gene 3000 e Eppendorf Mastercycler ep realplex e Roche LightCycler 480 e Stratagene Mx3000P Mx3005P and Mx40009 EXPRESS One Step SYBR GreenER with Premixed ROX can be used with real time instruments that are compatible with ROX Reference Dye at a final concentration of 500 nM These include the following Applied Biosystems instruments e 7900HT e 7300 e StepOne e StepOnePlus e GeneAmp 5700 e PRISM 7000 and 7700 Methods General qRT PCR Guidelines and Parameters Reaction Setup and Conditions Primer Specifications Melting Curve Analysis e Starting material can be total RNA or mRNA e These kits use a two step cycling protocol with a denaturation step at 95 C and an annealing extension step at 60 C e Keep all components reaction mixes and samples on ice to prevent premature cDNA synthesis e Reaction volumes can be scaled from 5 pl to 100 ul depending on the instrument e For most templates efficient cDNA synthesis can be accomplished in a 5 minute incuba
26. ued on next page Template RNA continued Determining Total RNA Yield Determining Total RNA Quality Total RNA can be quantitated using the Quant iT RNA Assay Kit or UV absorbance at 260 nm Quant iT RNA Assay Kit The Quant iI RNA Assay Kit provides a rapid sensitive and specific method for RNA quantitation with minimal interference from DNA protein or other common contaminants that affect UV absorbance readings The kit contains a quantitation reagent and pre diluted standards for a standard curve The assay is performed in a microtiter plate and can be read using a standard fluorescent microplate reader UV Absorbance 1 Dilutean aliquot of the total RNA sample in 10 mM Tris HCl pH 7 5 Mix well Transfer to a cuvette 1 cm path length Note The RNA must be in a neutral pH buffer to accurately measure the UV absorbance 2 Determine the OD o of the solution using a spectrophotometer blanked against 10 mM Tris HCl pH 7 5 Calculate the amount of total RNA using the following formula Total RNA ug OD o x 40 ug 1 OD260 x 1 ml x dilution factor x total sample volume ml Example Total RNA was eluted in water in a total volume of 150 ul A 40 l aliquot of the eluate was diluted to 500 ul in 10 mM Tris HCl pH 7 5 An OD e of 0 188 was obtained The amount of RNA in the sample is Total RNA ug 0 188 x 40 ug 1 OD o x 1 ml x 12 5 x 0 15 14 1 ug Total RNA quality can be analyzed u
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