User Manual - Biomol GmbH
Contents
1. 1 2 3 Additional Materials Required RT qPCR Grade miRNA Isolation Kit Cat No MA 01 RT miRNA First Strand Kit Cat No MA 03 MANDATORY for a Complete and Successful Experiment The universal primer in each assay is only compatible with the sequence added to the cDNA template by the primer in this kit SABiosciences RT SYBR Green qPCR Master Mix MANDATORY for a Complete and Successful Experiment Be sure to pick the correct one for the instrumentation in your laboratory RT Real Time SYBR Green ROX Specifically designed for All ABI and Stratagene Instrumentation and Eppendorf Mastercycler ep realplex Instruments with ROX filter set Catalog Number Size PA 012 For 200 25 uL reactions 2 5 mL PA 112 For 2000 25 uL reactions 25 0 mL RT Real Time SYBR Green Fluorescein Specifically designed for BioRad iCylcer MyiQ and iQ5 Instrumentation Catalog Number Size PA 011 For 200 25 uL reactions 2 5 mL PA 111 For 2000 25 uL reactions 25 0 mL RT Real Time SYBR Green Specifically designed for instrumentation that does not require a reference dye BioRad MJ Research Opticon Opticon 2 and Chromo 4 Roche LightCycler 480 System Eppendorf Mastercycler ep realplex Instruments without ROX filter set Catalog Number Size PA 011 For 200 25 uL reactions 2 5 mL PA 111 For 2000 25 uL reactions 25 0 mL Equipment For recommendations on specific real time instrumentation2. C value lt 35 1 DNA contamination of other reagents tips and tubes See the Note on Preparing a Workspace Free of DNA Contamination at the beginning of the protocol in this User Manual 2 Primer Dimers Verify presence of primer dimers lt 50 bp in size by agarose gel Be sure to use the appropriate RT2 qPCR Master Mix from SABiosciences to prevent the appearance of primer dimers B Frequently Asked Questions 1 Will pipetting error affect the miRNA qPCR Assay results The passive reference dyes in the PCR master mixes such as ROX and Fluorescein are used by the real time PCR systems to normalize variation from well to well Therefore these systems tolerate volume variations caused by pipetting error and evaporation 2 How can prevent the evaporation of reaction volume from the wells Be sure to carefully and completely seal the PCR plate with the optical thin wall 8 cap strips or the optical adhesive film before placing it into your thermal cycler Also be sure to use a compression pad when using an optical film seal as directed by the manufacturer of your real time PCR instrument 3 How reliable are the results from the miRNA qPCR Assays Assuming the use of good consistent experimental technique real time PCR methods such as the miRNA qPCR Assays provide very reproducible results To insure the reliability of your results and to reliably detect smaller fold changes in miRNA expression from the PCR Assays the per
3. Poor quality RNA can inhibit enzyme activity during reverse transcription generating an insufficient amount of template during the reverse transcription reaction 3 Not enough template a b Use more input RNA for reverse transcription especially if the lower end of the recommended range had been used previously Use a larger volume of template per reaction but do not use more than 2 0 ul of template per 25 ul reaction Remember to use the same volume of template in each reaction C Real time PCR Ct values are too low lt 12 Too much template 1 2 Use less input RNA for reverse transcription especially if the higher end of the recommended range had been used previously Use a smaller volume of template per reaction or use diluted template but do not use less than 1 ul of template per each 25 ul reaction Remember to use the same volume of template in each reaction 18 Version 1 4 D Expression is seen when it is not expected OR The No Reverse Transcription Control NRT yields a C value lt 35 If total RNA instead of small RNA is used in the miRNA qPCR more non specific products and higher background are expected In this case using small RNA will normally resolve the problem Be sure to use the RT qPCR Grade miRNA Isolation Kit MA 01 to enrich for small RNA from your biological samples of total RNA samples previously purified with the TRIZol procedure E The No Template Control NTC yields a
4. miRNA qPCR Assays is specific only for the unique and proprietary sequence incorporated into the cDNA by the universal RT primer in SABiosciences s RT miRNA First Strand Kit The miRNA gPCR Assays cannot detect cDNA generated from miRNA using other sources of miRNA first strand synthesis or miRNA reverse transcription kits Similarly CDNA generated from SABiosciences s RT miRNA First Strand Kit cannot be used with other sources of real time qPCR assays for miRNA The RT miRNA First Strand Kit also includes a proprietary buffer to preferentially reverse transcribe miRNA into cDNA The buffer components and the magnesium concentration in our RT miRNA First Strand Kit are also more compatible with our PCR master mixes than other enzymes or kits providing the miRNA qPCR Assays with maximum levels of sensitivity with ng amounts of small RNA Technical Support 888 503 3187 US 301 682 9200 7 miRNA qPCR Assays NOTE Preparing a Workspace Free of DNA Contamination For accurate and reproducible PCR Assay results it is very important to avoid contamination of the assay with foreign DNA Any DNA contamination will artificially inflate the SYBR Green signal yielding skewed expression profiles and false positive signals The most common sources of DNA contamination are the products of previous experiments spread into the air of your working environment Please follow the recommendations below on how to set up and maintain a working environment fr
5. thermal cyclers with fluorescent detection see the list of master mixes and plate formats above Calibrated Pipettors RNase DNase free pipette tips and tubes Version 1 4 IV Protocol Please read through this entire protocol before beginning your experiment RNA samples are very sensitive to RNase digestion therefore wear gloves and maintain an RNase free work area while performing this protocol NOTE Master Mix and Reverse Transcription Kit Considerations The performance of our miRNA qPCR Assays is only guaranteed with our RT SYBR Green qPCR Master Mixes and the RT miRNA First Strand Kit Therefore the use of the complete miRNA qPCR Assay System is absolutely essential for obtaining any real time PCR profiling results The chemically modified and tightly controlled HotStart enzyme and other proprietary chemical components in our RT SYBR Green qPCR Master Mixes provide more accurate SYBR Green results by preventing the amplification of primer dimers and other non specific products They also help insure high amplification efficiencies When we test other sources of enzymes with our miRNA qPCR Assays we frequently see primer dimers and other non specific products that confound SYBR Green based real time PCR detection Because each instrument uses a different reference dye to normalize their optics be sure that you use the correct master mix for the instrumentation in your laboratory The universal primer in every assay of the
6. to amplify a single amplicon with uniform PCR efficiency e Complete Genome Coverage Assays are available for over 400 majority miRNA sequences The uniform PCR efficiency and conditions allow easy scalable transitions from analyzing single to multiple miRNA sequences e Simple amp Convenient Universal reverse transcription converts all miRNA into cDNA in a one step reaction Assays deliver guaranteed performance when used with SABiosciences s optimized instrument specific qPCR master mixes for ABI Bio Rad Stratagene and other real time PCR instruments Version 1 4 How miRNA PCR ARRAYS WORK 1 Convert miRNA to cDNA Wi J Polyadenylation pi om 3 eae Reverse Transcription from Universal RT Primer 5p amm 3 Ga 5 Uni RT Prir 2 Add cDNA to RT qPCR Master Mix Aliquot Mixture Across PCR Array qPCR using miRNA Specific Primer and Universal qPCR Primer 3 Run in Your Real Time PCR Instrument Profile 1 Profile 2 Figure 1 Overview of the miRNA qPCR Assay procedure Technical Support 388 503 3187 US 301 682 9200 5 miRNA qPCR Assays Il Materials Provided RT miRNA qPCR Assay one tube 200 reaction scale Primer pair includes one miRNA specific primer and one universal primer One Product Specification Sheet Storage Conditions Primer Sets are shipped at ambient temperature but must be stored at 20 C where they are guaranteed as is for 6 months from the date received A B C D
7. 22769 Hamburg info biomol de J saBiosciences wet CY biomol U S e r M a n u a Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D RT miRNA qPCR Assays Real Time PCR Based miRNA Expression Profiling See Purchaser Notification for limited use license and warranty information page 3 Part 1032A Version 1 4 03 02 2009 SABiosciences Corporation 6951 Executive Way Frederick MD 21703 USA RT miRNA qPCR Assays Real Time PCR Based miRNA Expression Profiling User Manual For Catalog Number Prefix MPH MPM Ordering and Technical Service Contact Information BIOMOL GmbH e Tel 1 888 503 3187 US 301 682 9200 outside US 22769 Hamburg O biomol e Fax 1 888 465 9859 US 301 682 7300 outside US Www biomolde e On line Order www sabiosciences com Fa a asa a302 000 2 CECA D e E MAIL order sabiosciences com to place an order support sabiosciences com for technical support You may place orders by fax e mail or from our website Each order should include the following information Your contact information name phone email address Product name catalog number and quantity Purchase order number or credit card information Visa or MasterCard Shipping address Billing address For more information visit us at www sabiosciences com SABioscience Corporation 7320 Executive Way Suite 101 Frederick MD 21704 USA CONTENTS l Background and Introduction 4 Il M
8. OI relative expression levels by the corresponding HK relative expression levels Use MOI and HK data from the same dilution of the same sample Fold Changes To determine the fold change in expression for each miRNA sequence of interest between two different samples calculate the ratio of its normalized expression levels again determined from the same PCR conditions between those samples Technical Support 888 503 3187 US 301 682 9200 17 miRNA qPCR Assays VI Troubleshooting and FAQs A Appearance of multiple PCR products dissociation curve peaks 1 Assay Primers Amplify from Non miRNA cDNA If total RNA instead of small RNA is used in the miRNA qPCR more non specific products and higher background are expected In this case using small RNA will normally resolve the problem Be sure to use the RT qPCR Grade miRNA Isolation Kit MA 01 to enrich for small RNA from your biological samples of total RNA samples previously purified with the TRIzol procedure If the raw C value from the assay is greater than 35 consider the miRNA as Not Detectable and discounted B Real time PCR C values are too high gt 35 or not detectable 1 Experimental error Use cDNA template generated from the synthetic miRNA sequence of interest as a positive control to troubleshoot the PCR reagents and experimental procedure Poor RNA quality Be sure to perform all recommended quality control checks on the RNA sample
9. Results from the miRNA qPCR Array Displayed is an Agilent BioAnalyzer electropherogram of a high quality small RNA preparation showing a sharp low molecular weight peak without a shoulder 3 Amount Considerations The miRNA qPCR Assay System yields relative miRNA expression profiles with as little as 1 ng or as much as 200 ng of small RNA However the optimal amount of starting material depends upon the relative abundance of the sequences of interest Lower abundance sequences require more RNA higher abundance sequences require less RNA Greater amounts of input RNA yield a greater number of positive calls that is sequences expressed in the linear dynamic range of the method Lower amounts of input RNA yield a smaller number of positive calls and increase false negative calls The use of the RT miRNA First Strand Kit maximizes the number of positive calls at low amounts of RNA over other sources of miRNA reverse transcriptase and first strand synthesis kits For successful results and maximum positive call rates we recommend that first time users try starting with 100 ng of small RNA It is also important to use a consistent amount of RNA for each sample to be characterized and compared 10 Version 1 4 B RT miRNA First Strand Kit NOTE RNA samples must meet the standards of integrity and purity from protein and organics contamination described on the previous two pages NOTE The buffer components of the reverse transcripti
10. aterials Provided 6 III Additional Materials Required 6 IV Protocol 7 A RNA Preparation and Quality Control 9 B RT miRNA First Strand Kit 11 C Performing Real Time PCR 12 D Data Analysis AAC Method 15 E Data Analysis Optional Standard Curve Method 17 VI Troubleshooting and Frequently Asked Questions 18 LIMITED PRODUCT WARRANTY This product is intended for research purposes only and is not intended for drug or diagnostic purposes or for human use This warranty limits our liability to replace this product in the event the product fails to perform due to any manufacturing defect SABioscience Corporation makes no other warranties of any kind expressed or implied including without limitation warranties of merchantability or fitness for a particular purpose SABioscience Corporation shall not be liable for any direct indirect consequential or incidental damages arising out of the use the results of use or the inability to use this product NOTICE TO PURCHASER II The purchase of RT miRNA qPCR Assay includes a limited nonexclusive license to use the kit components for research use only This license does not grant rights to use the kit components for reproduction of any primer pair mix to modify kit components for resale or to use RT miRNA qPCR Assay to manufacture commercial products without written approval of SABioscience Corporation No other license expressed implied or by estoppels is granted U S patents may cover certai
11. ee of DNA contamination 1 Wear gloves throughout the procedure Use only fresh PCR grade reagents H20 and lab ware tips and tubes 2 Physically separate the workspaces used for PCR setup and post PCR processing or non PCR operations Decontaminate your PCR workspace and lab ware pipettor barrels tube racks etc before each new use with UV light to render any contaminating DNA ineffective in PCR through the formation of thymidine dimers or with 10 bleach to chemically inactivate and degrade any DNA 3 Close all tubes containing PCR products once you are finished adding or removing volumes Before discarding any lab ware tips or tubes containing PCR products or other DNA treat with 10 bleach 4 Do not open any previously run and stored PCR plate Removing the thin wall 8 cap strips or the adhesive film from PCR plates releases PCR product DNA into the air where it will contaminate and confound the results of future real time PCR experiments Version 1 4 A RNA Preparation and Quality Control High quality RNA is ESSENTIAL for obtaining good real time PCR results The most important prerequisite for any miRNA expression analysis experiment is consistent high quality small RNA from every experimental sample Therefore the sample handling and RNA isolation procedures are critical to the success of the experiment Residual traces of proteins salts or other contaminants will either degrade the RNA or decrease the efficiency o
12. evel of a miRNA of interest GOI to a housekeeping sequence HKG the expression levels of the two miRNA sequences are divided g CiGOl Sn C G01 C HKG _ 4 AC 9 canKG 2 To determine fold change in miRNA expression the normalized expression of the GOI in the experimental sample is divided by the normalized expression of the same GOI in the control sample 9 Shiiexnt yabeariety 2 Where AAC is equal to AC expt AC control The complete calculation is as follows grAC GON expt ZACKS expt 2 IC4GON CHKO oyp _ 2 expt _ hae 226 control 21460 HKG Control 2 control J AGIHKS control 16 Version 1 4 E Data Analysis Optional Standard Curve Method 1 Raw Data Determine the threshold cycle C value for each reaction using your instrument s software Generate a standard curve for each MOI and each HK assay Plot C values of the assays using synthetic miRNA templates on the y axis versus their dilution factors in a log scale on the x axis Fit the data to a straight line The x axis value is directly related to the relative expression level L of the miRNA sequence Determine Relative Expression of Unknowns Use the standard curve or linear fit equation to convert C values to relative expression levels for each MOI or HK sequence in each sample Be sure that the C values fall within the linear range of the appropriate standard curve Normalization Divide each M
13. f if not block completely the enzyme activities necessary for optimal reverse transcription and real time PCR performance Although other real time PCR based miRNA detection methods call for the use of total RNA that contains miRNA as the starting input material we highly recommend starting with small RNA lt 200 nucleotides to reduce background noise for optimal sensitivity 1 Recommended RNA Preparation Methods High quality small RNA for your real time PCR experiment must be prepared using one of the following methods each specific for your biological sample Cultured Cells OR Tissue Samples Use a two step protocol a Extract RNA from the tissue using the TRIzol protocol Invitrogen Catalog 15596 026 Be sure to use a sufficient amount of TRIzol reagent During homogenization add a volume of reagent at least ten times greater than the tissue volume b Then further clean up and enrich the small RNA using the RT gPCR Grade miRNA Isolation Kit SABiosciences MA 01 Whole Blood Samples Remove red blood cells RBC from whole blood samples using a density gradient centrifugation medium for example Lymphoprep Greiner Bio One Catalog 1031966 Isolate small RNA from the white blood cell fraction as described for cultured cells above Previously Isolated Total RNA If you have already prepared total RNA from any biological source material be sure that it used a phenol based method such as TRIzol RNAzol e
14. formance of replicate determinations such as biological triplicates is highly recommended If you have additional questions please check our website www sabiosciences com for a more complete listing of Frequently Asked Questions FAQs or call our Technical Support Representatives at 1 888 503 3187 or 301 682 9200 iCycler and MyiQ are registered trademarks of BioRad Laboratories Inc LabChip is a registered trademark of Caliper Life Sciences LightCycler is a registered trademark of Roche Applied Sciences RNeasy is a registered trademark of Qiagen SmartCycler is a registered trademark of Cepheid SYBR is a registered trademark of Molecular Probes TRIzol is a registered trademark of Invitrogen Mastercycler is a registered trademark of Eppendorf Technical Support 888 503 3187 US 301 682 9200 19 miRNA qPCR Assays miRNA gPCR Assay User Manual Part 1032A Version 1 4 03 02 2009 fy SABiosciences Focus on Your Pathway BIOMOL GmbH Waidmannstr 35 O m 22769 Hamburg b m info biomol de 10 o www biomol de Phone 49 40 8532600 or 0800 2466651 D Fax 49 40 85326022 or 0800 2466652 D 888 503 3187 301 682 9200 www SABiosciences com support SABiosciences com 20
15. from the miRNA PCR Assays Be sure to use the correct master mix for your instrument before continuing with this protocol See Pages 8 and 10 NOTE The accuracy and precision of your pipetting determines the consistency of your results Be sure that all of your micro pipettors are calibrated before beginning this procedure Also make sure to not introduce any bubbles into the PCR wells NOTE f unsure of your RNA quality or isolation technique examine the quality of your RNA before this step 1 Setup a Experimental Samples To insure the consistency of your results and that each experimental sample yields a reliably detectable C value we recommend using undiluted template and a 1 10 dilution of your template in separate PCR assays Also prepare triplicate reactions for each template at each concentration For every experimental sample prepare one set of reactions for every miRNA sequence of interest and for a single housekeeping sequence or a set of housekeeping sequences to normalize your raw data Choose housekeeping sequence s such as small nuclear RNA species known not to change their expression under your experimental conditions b Positive and Negative Controls Prepare a positive control reaction for each miRNA qPCR Assay using cDNA template generated by reverse transcribing 2 uL of a 1 10 nM solution of synthetic RNA oligo with the same sequence s as the miRNA of interest To control for DNA contamination introd
16. g the instrument manufacturer s instructions NOTE Be sure that no bubbles appear in any of the wells of the PCR plate To remove bubbles tap the plate gently on the bench top or centrifuge the plate briefly b Place the plate on ice while setting up the PCR cycling program below c Place the plate in your real time thermal cycler If recommended by your instrument s user manual use a compression pad on plates sealed with optical film d Enter and run the appropriate program for your real time instrument below If prompted by your instrument software select Absolute Quantitation to begin NOTE For additional help with instrument setup see our Instrument Specific Setup Instructions and Protocol Files at www sabiosciences com pcrarrayprotocolfiles php Cycles Duration Temperature 1 10 minutes 95 C 15 seconds 95 C 40 30 to 40 seconds 60 C 30 seconds 72 C 1 The 10 minute step at 95 C is required to activate the HotStart DNA polymerase Detect and record SYBR Green fluorescence from every well during the annealing step of each cycle Different instruments need different lengths of time to detect the fluorescent signal Choose the appropriate time for the annealing step 60 C for your instrument Technical Support 888 503 3187 US 301 682 9200 13 miRNA qPCR Assays e Calculate the threshold cycle C for each well using the instrument s software i We highly recommend letti
17. ion A raw C value less than 35 indicates the presence of general DNA contamination in the PCR See the Troubleshooting section of this User Manual and the Note on Preparing a Workspace Free of DNA Contamination earlier in this protocol 3 Calculate the AC for each miRNA in each sample and each dilution ACi ee SG Ae HKG 4 When biological and or technical replicates are performed calculate the average AC value of each miRNA assay across those replicates for each treatment group 5 Calculate the AAC for each miRNA between your two PCR samples or groups AAC AC group 2 AC group 1 Where group 1 is the control and group 2 is the experimental 6 Calculate the fold change for each miRNA from group 1 to group 2 as 2 AAC OPTIONAL f the fold change is greater than 1 then the result may be reported as a fold up regulation If the fold change is less than 1 then the negative inverse of the result may be reported as a fold down regulation The fold change ratios may also be reported as is Technical Support 888 503 3187 US 301 682 9200 15 miRNA qPCR Assays NOTE Detailed Mathematical Explanation of AAC Data Analysis Method Due to the inverse proportional relationship between the threshold cycle C and the original miRNA expression level and the doubling of the amount of product with every cycle the original expression level L for each miRNA of interest is expressed as Ler To normalize the expression l
18. n isolated DNA sequences included in the RT miRNA qPCR Assay Presently it is not clear under U S laws whether commercial users must obtain licenses from the owners of the rights to these U S patents before using RT miRNA qPCR Assay I Background and Introduction MicroRNA miRNA first discovered in C elegans in early 1990s are a class of naturally occurring small RNA molecules generally 17 to 30 nucleotides long processed from much larger stem loop structures pri miRNA and pre miRNA Mature miRNA is recognized by and guides the RNA Induced Silencing Complex RISC to specific messenger RNA transcript sequences causing either translation repression or MRNA degradation The expression and function of miRNA brings another layer of gene expression regulation to an already complex network Each miRNA can regulate multiple mRNA targets and a single target MRNA may be regulated by multiple miRNA sequences Researchers are currently correlating miRNA expression profiles to biological phenotypes in attempts to better understand miRNA based gene regulation Real time reverse transcription PCR or qRT PCR is the most sensitive and reliable method for nucleic acid expression analysis However the small size and high degree of similarity among miRNA sequences makes quantitative analyses of their abundance very challenging The RT miRNA gPCR Assays take advantage of SABiosciences s patent pending primer design and assay formulation technologie
19. ng your instrument automatically define the Baseline values but manually setting the Threshold values ii To define the Baseline use the Linear View of the amplification plots and set the instrument to use the readings from cycle number two 2 through two 2 cycle values before the earliest visible amplification usually around cycle number ten 10 but no more than 15 iii To define the Threshold Value use the Log View of the amplification plots and place it above the background signal but within the lower half to one third of the linear phase of the amplification plot iv IMPORTANT Ensure that the Thresholds are the same across all PCR runs in the same analysis v Export the resulting threshold cycle values for all wells to a blank Excel spreadsheet for use with our Data Analysis Template Excel file 4 Recommended Quality Control a Dissociation Melting Curve Run a melting curve program immediately after the above cycling program and generate a first derivative dissociation curve for each well in the entire plate using your instrument s software No more than one peak should appear in each reaction at temperatures greater than 70 C If your instrument does not have a default melting curve program run the following program instead 95 C 1 min 65 C 2 min OPTICS OFF 65 C to 95 C at 2 C min OPTICS ON If you decide not to obtain the dissociation curve immediately save the plates wrapped in aluminum f
20. oil at 20 C as is in case you need to perform this operation at a later point in time for troubleshooting purposes When ready simply warm the plate to room temperature place it into your real time instrument and run the melting program described above NOTE Be sure to visually inspect the plate after the run for any signs of evaporation from any of the wells If evaporation is observed make a note of which wells so that you may qualify your data analysis appropriately NOTE DO NOT open any previously run and stored PCR plate Removing the thin wall 8 cap strips or the adhesive film from PCR plates releases PCR product DNA into the air where it will contaminate and confound the results of future real time PCR experiments See also the Note on Preparing a Workspace Free of DNA Contamination 14 Version 1 4 D Data Analysis AAC Method 1 Change all C values reported as greater than 35 or as N A not detected to 35 At this point any C value equal to 35 is considered a negative call 2 Examine the threshold cycle values of the control wells a Positive Control b No Reverse Transcription NRT A raw C value of 35 or greater indicates a lack of non specific background A raw C value less than 35 indicates the presence of secondary amplification products See the Troubleshooting section of this User Manual c No Template Control NTC A raw C value of 35 or greater indicates a lack of general DNA contaminat
21. on reaction using this kit are more compatible with our PCR master mixes than other sources of kits for first strand cDNA synthesis kits or reverse transcriptases NOTE Thaw component M2 on ice briefly vortex the tube to mix the contents well and centrifuge to the bottom of the tube before each use For each RNA sample combine the following in a sterile PCR tube Small RNA 50 ng to 200 ng or 0 5 2 ug total RNA miRNA RT Primer amp ERC Mix M1 1 0 uL 5X miRNA RT Buffer M2 2 0 uL miRNA RT Enzyme Mix M3 1 0 uL 100 mM DTT 1 0 uL RNase free H20 to a final volume of 10 0 uL NOTE Use the same amount of RNA in this reaction for every sample First time users are recommended to start with 100 ng of small RNA Do not use more than 400 ng of small RNA per reaction b Mix the contents gently with a pipettor followed by brief centrifugation c Incubate at 37 C for 2 hours d Heat at 95 C for 5 minutes to degrade the RNA and to inactivate the reverse transcriptase e Chill on ice for at least one minute and add 90 ul of RNase free H20 to each 10 nl of cDNA synthesis reaction Mix well f Hold the finished First Strand cDNA Synthesis Reaction on ice until the next step or store overnight at 20 C Technical Support 888 503 3187 US 301 682 9200 11 miRNA qPCR Assays C Performing Real Time PCR NOTE The use of SABiosciences s RT SYBR Green qPCR Master Mixes is critical for obtaining the most accurate results
22. s to analyze the expression of any mature miRNA sequence in the human or mouse genome by SYBR Green based real time RT PCR The assays rely on combining poly A tailing and a universal reverse transcription in one first strand cDNA synthesis reaction Our proprietary primer design algorithm also distinguishes miRNA family members with single nucleotide mismatches Each assay is experimentally validated with an in vitro assay to insure the amplification of a single product with at least consistently high efficiency under standardized cycling conditions The high performance of every RT miRNA qPCR Assay is certified and 100 guaranteed when performed using the complete miRNA PCR Assay System that includes the RT miRNA First Strand Kit and RT SYBR Green qPCR Master mix To complete the miRNA PCR Assay procedure reverse transcribe your experimental small RNA samples into first strand cDNA the template for the PCR using our RT miRNA First Strand Kit See Figure 1 for an overview of the miRNA PCR Assay procedure Then mix the template with one of our instrument specific and ready to use RT SYBR Green qPCR Master Mixes Set up your assays perform PCR and finally determine relative expression with your real time instrument and the AAC method The simplicity of the miRNA qPCR Assays makes them accessible for routine use in every research laboratory Benefits of the RT miRNA qPCR Assays e High Peformance Each assay is experimentally validated
23. tc Then enrich for small RNA using the RT gPCR Grade miRNA Isolation Kit SABiosciences MA 01 For Other Biological Samples Refer to the existing literature to find isolation protocols for high quality RNA from other biological samples or contact one of our Technical Support representatives For best results from the PCR Assays all RNA samples should be suspended in the RNase free water provided with the RNA Isolation kit or alternatively in RNase free 10 mM Tris buffer pH 8 0 DO NOT use DEPC treated water Technical Support 888 503 3187 US 301 682 9200 9 miRNA qPCR Assays 2 RNA Quality Control For best results from the PCR Assays all RNA samples should also demonstrate consistent quality according to the following criteria a RNA Concentration and Purity by UV Spectrophotometry NOTE Prepare dilutions and measure absorbance in 10 mM Tris pH 8 0 buffer The spectral properties of nucleic acids are highly dependent on pH i A260 A230 ratio should be greater than 1 7 ii A260 A280 ratio should be greater than 2 0 iii Concentration by Azgo should be greater than 1 ug ml small RNA b RNA Quality amp Integrity Characterize 10 ng of the small RNA on an Agilent Bioanalyzer using an RNA 6000 Nano LabChip The RNA should contain a single sharp peak at a low molecular weight with no smearing and no additional peaks at higher molecular weights Figure 3 Good Small RNA Band Integrity Is Important for Best
24. uced during reaction setup prepare a negative control reaction replacing template with water the so called No Template Control NTC To monitor non specific background amplification perform one assay for each miRNA and each housekeeping sequence of interest using an equivalent volume of product from a reaction replacing the miRNA RT Enzyme Mix M3 with RNase free H20 for each miRNA sample the so called No Reverse Transcription NRT control c Optional Standard Curve Method Generate one standard curve for each miRNA of interest Also generate a standard curve for each housekeeping small nuclear RNA sequence to be used for data normalization To generate a standard curve prepare a five point series of five or ten fold dilutions in duplicate using cDNA template generated by reverse transcribing synthetic miRNA with the sequence s of interest 12 Version 1 4 2 Polymerase Chain Reactions For each 25 ul PCR mix the following components in a PCR tube 12 5 ul RT Real Time PCR master mix matched with your instrument 10 5 ul ddH20 1 0 ul of either undiluted or diluted template 1 0 ul RT2 miRNA qPCR Assay primer set 25 0 ul final volume 3 Performing Real Time PCR Detection NOTE Be sure to follow the manufacturer s instructions for the proper operation and maintenance of your real time instrument a CAREFULLY but tightly seal the PCR plate either with optical thin wall 8 cap strips or optical adhesive film accordin
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