GoScript Reverse Transcription System Protocol

T e c h n i c a l M a n u a l GoScript™ Reverse Transcription System INSTRUCTIONS FOR USE OF PRODUCTS A5000 AND A5001. PRINTED IN USA. Revised 12/12 Part# TM316 tm316.1212:EIVD_TM.qxd 12/18/2012 8:29 AM Page a Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Part# TM316 Revised 12/12 Page 1 1. Description ..........................................................................................................1 2. Product Components and Storage Conditions ............................................2 3. General Laboratory Precautions .....................................................................3 4. Detailed Protocol ...............................................................................................6 A. First-Strand cDNA Synthesis..............................................................................7 B. cDNA Quantification Using qPCR ....................................................................8 C. PCR Amplification for Endpoint Analysis .......................................................9 D. Analysis................................................................................................................11 5. Protocol Optimization.....................................................................................11 A. GoScript™ Reverse Transcription System Reaction Characteristics ..........11 B. RNA Template ....................................................................................................12 C. Magnesium Concentration Optimization.......................................................12 D. Primer Options and Design ..............................................................................13 E. Reverse Transcriptase Enzyme Concentration ..............................................13 F. Control Reactions ...............................................................................................13 G. Temperature ........................................................................................................14 6. Troubleshooting...............................................................................................15 7. References .........................................................................................................20 8. Related Products ..............................................................................................20 1. Description The GoScript™ Reverse Transcription System(a,b) is a convenient kit that includes a reverse transcriptase and an optimized set of reagents designed for efficient synthesis of first-strand cDNA optimized in preparation for PCR amplification. The components of the GoScript™ Reverse Transcription System can be used to reverse transcribe RNA templates starting with total RNA, poly(A)+ mRNA or synthetic transcript RNA. Figure 1 provides an overview of the reverse transcription procedure. The optimized reaction buffer and reverse transcriptase provided in the GoScript™ Reverse Transcription System enable robust, full-length cDNA synthesis for reproducible analysis of rare or long messages. GoScript™ Reverse Transcription System All technical literature is available on the Internet at: www.promega.com/protocols/ Please visit the web site to verify that you are using the most current version of this Technical Manual. Please contact Promega Technical Services if you have questions on use of this system. E-mail: techserv@promega.com tm316.1212:EIVD_TM.qxd 12/18/2012 8:29 AM Page 1 1. Description (continued) These conditions were developed for cDNA synthesis or for easy transition to gene-specific target amplification. Up to 5µl of the reverse transcription reaction can be directly amplified using Taq DNA polymerase in a 25µl PCR. GoScript™ Reverse Transcriptase is qualified for use in two-step RT-qPCR using GoTaq® qPCR and Plexor® qPCR Systems. In two-step RT-qPCR using GoTaq® qPCR Master Mix, samples of the GoScript™ Reverse Transcription System can be added directly, up to 20% v/v. 2. Product Components and Storage Conditions Product Size Cat.# GoScript™ Reverse Transcription System 50 reactions A5000 Each system contains sufficient reagents for 50 first-strand cDNA synthesis reactions of 20µl each. • 50µl GoScript™ Reverse Transcriptase • 300µl GoScript™ 5X Reaction Buffer • 750µl MgCl2 (25mM) • 200µl PCR Nucleotide Mix • 50µg Oligo(dT)15 Primer • 50µg Random Primers • 1.25ml Nuclease-Free Water • 2,500u Recombinant RNasin® Ribonuclease Inhibitor Product Size Cat.# GoScript™ Reverse Transcription System 100 reactions A5001 Each system contains sufficient reagents for 100 first-strand cDNA synthesis reactions of 20µl each. • 100µl GoScript™ Reverse Transcriptase • 600µl GoScript™ 5X Reaction Buffer • 1.2ml MgCl2 (25mM) • 200µl PCR Nucleotide Mix • 50µg Oligo(dT)15 Primer • 50µg Random Primers • 1.25ml Nuclease-Free Water • 2,500u Recombinant RNasin® Ribonuclease Inhibitor Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TM316 Printed in USA. Page 2 Revised 12/12 tm316.1212:EIVD_TM.qxd 12/18/2012 8:29 AM Page 2 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Part# TM316 Revised 12/12 Page 3 Available Separately Product Size Cat.# GoScript™ Reverse Transcriptase 10 reactions A5002 GoScript™ Reverse Transcriptase 100 reactions A5003 GoScript™ Reverse Transcriptase 500 reactions A5004 Storage Conditions: Store all system components at –20°C. Thaw and maintain the GoScript™ 5X Reaction Buffer, the GoScript™ Reverse Transcriptase and PCR Nucleotide Mix on ice during use. See the expiration date on the system label. 3. General Laboratory Precautions • Use designated work areas and pipettors for pre- and post-amplification steps. This precaution is intended to minimize the potential for cross-contamination between samples and prevent carryover of nucleic acid (DNA and RNA) from one experiment to the next. • Wear gloves and change them often. • Prevent contamination by using barrier or positive displacement pipette tips. • Use sterile, nuclease-free thin-walled reaction tubes. • The GoScript™ Reverse Transcriptase, GoScript™ 5X Reaction Buffer and PCR Nucleotide Mix should be kept chilled before use. Thaw on ice; do not thaw by heating in a warming block. Note: The characteristics of the products of the reverse transcriptase reaction run in the 37–55°C temperature range may vary, depending on the RNA template and the method of analysis of the products. tm316.1212:EIVD_TM.qxd 12/18/2012 8:29 AM Page 3 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TM316 Printed in USA. Page 4 Revised 12/12 Oligo (dT)15 Primer Random Primer Heat to 70°C for 5 minutes. Quick chill on ice for 5 minutes. Prepare reverse transcription mix. Dispense into aliquots and add primer/RNA mix. Anneal at 25°C for 5 minutes.(1) Carry out first-strand synthesis reaction 42°C(2) for 60 minutes. ANALYSIS Gene-Specific Primer 5´ AAAAA TTTTT–5´ 5´ AAAAA TTTTT–5´ 5´ NNNNNN–5´ 5´ NNNNNN–5´ 5´ GATCGATC–5´ 5´ GATCGATC–5´ AAAAA 5´ TTTTT–5´ NNNNNN–5´ 5´ 5´ GATCGATC–5´ (1) May optimize annealing temperature. (2) May optimize between 37°C and 55°C. Direct cDNA Analysis Gel electrophoresis, microarray, other Storage –20°C Two-Step RT-PCR Inactivation of reverse transcriptase (70°C for 15 minutes) Add sample of RT reaction to PCR or qPCR amplification. 32 93 M A0 3_ 1A Combine RNA and cDNA primer: and/or or Figure 1. Schematic overview of cDNA synthesis and downstream analysis options using the GoScript™ Reverse Transcription System. tm316.1212:EIVD_TM.qxd 12/18/2012 8:29 AM Page 4 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Part# TM316 Revised 12/12 Page 5 85 65 TA A. B. Figure 2. GoScript™ Reverse Transcriptase cDNA synthesis coupled with GoTaq® qPCR Master Mix can detect input template over a 9-log order dynamic range. Panel A. Ten RNA template mixes were assembled to model a series of tenfold differences of a specific RNA abundance (1 × 102 to 1 × 1011 copies of MS2 bacteriophage RNA or 0.2fg to 200ng) in a constant mass of 10ng of human total RNA. cDNAs for each RNA sample were prepared using GoScript™ Reverse Transcriptase plus oligo(dT) and random primer in parallel with minus-RT control reactions. Each reverse transcription reaction was diluted (1:10) for use as template in cDNA-specific qPCR. Replicate (n = 3) GoTaq® qPCR at each level of total-cDNA template, minus-RT control and no-template control (NTC) was performed using primers specific to MS2 cDNA. Semilog-scale amplification plots are shown on the left, including the results of the standard, “unknown”, minus-RT controls and NTC (undetected) reactions. Inset (Panel A) shows dissociation profile for the 50-, 500- and 5,000-copy standard and 5-copy detected levels of MS2 + cDNA and NTC reactions, demonstrating specificity. Standard values are defined as the number of copies of input MS2 bacteriophage RNA in each sample of diluted reverse transcription reaction: 5 × 109 down to 5 × 101 copies in 500pg of human total RNA. Standard curve shown in Panel B illustrates the quantitation and detection of the “unknown” samples. • = 10-fold standard dilutions; xx = detection of 5-copy samples. tm316.1212:EIVD_TM.qxd 12/18/2012 8:29 AM Page 5 4. Detailed Protocol Materials to Be Supplied by the User • commercially autoclaved, nuclease-free, thin-walled reaction tubes, 0.5ml • sterile, aerosol-resistant tips and pipettors • high-quality, experimental target RNA diluted in nuclease-free water • ice-water bath • 25°C, 42°C and 70°C controlled-temperature water baths or heat blocks Options for subsequent amplification in RT-PCR include: • gene-specific primers for PCR priming • Taq DNA polymerase and appropriate reaction buffer or GoTaq® Hot Start Polymerase (Cat.# M5001) • PCR Nucleotide Mix and magnesium chloride, 25mM, or alternative amplification system such as GoTaq® Green Master Mix (Cat.# M7122) or GoTaq® Colorless Master Mix (Cat.# M5133) • qPCR system, i.e., GoTaq® qPCR Master Mix (Cat.# A6001) or Plexor® qPCR System (Cat.# A4011) This procedure outlines the synthesis of cDNA for subsequent amplification using PCR or qPCR. Reverse transcription reactions of up to 5µg of total RNA, poly(A)+ mRNA or synthetic transcript RNA are performed in 20µl reactions comprised of components of the GoScript™ Reverse Transcription System. Experimental RNA is combined with the experimental primer. The primer/template mix is thermally denatured at 70°C for 5 minutes and chilled on ice. A reverse transcription reaction mix is assembled on ice to contain nuclease-free water, reaction buffer, reverse transcriptase, magnesium chloride, dNTPs (PCR Nucleotide Mix) and ribonuclease inhibitor. In experimental systems, addition of 1u/µl of Recombinant RNasin® Ribonuclease Inhibitor is recommended but optional. As a final step, the template-primer combination is added to the reaction mix on ice. Following an initial annealing at 25°C for 5 minutes, the reaction is incubated at 42°C for up to one hour. Because no cleanup or dilution is necessary following cDNA synthesis, the product may be directly added to amplification reactions. This procedure outlines the method proposed to amplify up to a 5µl aliquot of the cDNA synthesis reaction product in 25µl PCR amplifications. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TM316 Printed in USA. Page 6 Revised 12/12 tm316.1212:EIVD_TM.qxd 12/18/2012 8:29 AM Page 6 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Part# TM316 Revised 12/12 Page 7 4.A. First-Strand cDNA Synthesis The following procedure is designed to convert up to 5µg of total RNA or up to 500ng of poly(A) RNA into first-strand cDNA. 1. Mix and briefly centrifuge each component before use. Combine the following: Component Experimental RNA (up to 5µg/reaction) Xµl Primer [Oligo(dT)15 (0.5µg/reaction) and/or Random Primer (0.5µg/reaction) or gene-specific primer (10–20pmol/reaction)] Xµl Nuclease-Free Water Xµl Final volume 5µl 2. Close each tube of RNA tightly. Place tubes into a preheated 70°C heat block for 5 minutes. Immediately chill in ice-water for at least 5 minutes. Centrifuge each tube for 10 seconds in a microcentrifuge to collect the condensate and maintain the original volume. Keep the tubes closed and on ice until the reverse transcription reaction mix is added. 3. Prepare the reverse transcription reaction mix by combining the following components of the GoScript™ Reverse Transcription System in a sterile microcentrifuge tube on ice. Prepare sufficient mix to allow 15µl for each cDNA synthesis reaction to be performed. Determine the volumes needed for each component, and combine them in the order listed. Vortex gently to mix, and keep on ice prior to dispensing into the reaction tubes. Component Amount Nuclease-Free Water (to a final volume of 15µl) Xµl GoScript™ 5X Reaction Buffer 4.0µl MgCl2 (final concentration 1.5–5.0mM)1 1.2–6.4µl PCR Nucleotide Mix (final concentration 0.5mM each dNTP)2 1.0µl Recombinant RNasin® Ribonuclease Inhibitor (optional) 20u GoScript™ Reverse Transcriptase 1.0µl Final volume 15.0µl 1Mg2+ concentration should be optimized. We recommend 1.5–5.0mM. (MgCl2 is provided at 25mM.) 2If isotopic or nonisotopic incorporation is desired to monitor this first-strand cDNA synthesis, α[32P]-dCTP or other modified nucleotides may be supplemented in the PCR Nucleotide Mix. See Section 4.D for analysis suggestions. tm316.1212:EIVD_TM.qxd 12/18/2012 8:29 AM Page 7 4.A. First-Strand cDNA Synthesis (continued) 4. Add 15µl aliquots of the reverse transcription reaction mix to each reaction tube on ice. Be careful to prevent cross-contamination. Add 5µl of RNA and primer mix to each reaction for a final reaction volume of 20µl per tube. If there is a concern about evaporation in subsequent steps, overlay the reaction with a drop of nuclease-free mineral oil to prevent evaporation and condensation. 5. Anneal: Place the tubes in a controlled-temperature heat block equilibrated at 25°C, and incubate for 5 minutes. 6. Extend: Incubate the tubes in a controlled-temperature heat block at 42°C for up to one hour. The extension temperature may be optimized between 37°C and 55°C. The reactions may be stopped at this point for cDNA analysis as outlined in Section 4.D. The reactions may be maintained frozen for long-term storage. 7. Inactivate Reverse Transcriptase: If the experimental goal is to proceed with PCR, the reverse transcriptase must be thermally inactivated prior to amplification. Incubate the reaction tubes in a controlled-temperature heat block at 70°C for 15 minutes. 4.B. cDNA Quantification Using qPCR cDNA synthesized using GoScript™ Reverse Transcriptase can be amplified and quantified using the GoTaq® qPCR Master Mix or Plexor® qPCR Systems. cDNA samples may be used directly or diluted prior to amplification. As a starting point for dilution, dilute sample and reference standard cDNA reactions 1:10, then add 5µl of these diluted reactions to the reaction mix. For additional information, refer to the GoTaq® qPCR Master Mix Technical Manual, #TM318, or the Plexor® qPCR System Technical Manual, #TM262. The synthesized cDNA may be added directly to PCR amplifications. Unlike other first-strand systems, there will be no inhibitory effects encountered when up to 20% of the reaction is added to a PCR amplification as long as the final MgCl2 concentration is kept at an optimal level. The robust reaction conditions of the GoScript™ Reverse Transcription System make many flexible applications possible. The method outlined in Section 4.C describes two-step RT-PCR using either 1µl or 5µl of the reverse transcription reaction in a 25µl PCR. The volumes of PCR components assembled take into account the carryover of buffer, magnesium and dNTP from the reverse transcription reaction to achieve the final concentration of each component. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TM316 Printed in USA. Page 8 Revised 12/12 tm316.1212:EIVD_TM.qxd 12/18/2012 8:29 AM Page 8 1. Heat inactivate the reverse transcription reaction. 2. Proceed directly to GoTaq® or Plexor® qPCR target-specific quantitative analysis of the cDNA. 3. Alternatively, heat-inactivated reactions may be stored frozen for future use. 4. GoTaq® and Plexor® qPCR accommodate addition of up to 20% of the total reaction volume as template GoScript™ Reverse Transcriptase reaction volume. 5. The cDNA may be added directly to the qPCR as undiluted reverse transcription reaction product, or it may be diluted. 6. The dilution factor must be experimentally determined to be appropriate for the amount of RNA template mass and proportional reverse-transcript representation in the cDNA sample. 7. Generally, for cDNA quantitation, using the default analysis settings of many real-time instruments, the amount of cDNA used as template in GoTaq® qPCR should not exceed that correlating with a proportional 100ng of input total RNA. The cDNA generated from highly abundant transcripts can be detected in less than 1pg of total RNA. 4.C. PCR Amplification for Endpoint Analysis Note: GoScript™ Reverse Transcription reaction conditions support PCR amplification. No dilution of the cDNA is necessary. Add heat-inactivated reverse transcription reaction products directly to the PCR mix. 1. The cDNA may be amplified directly by adding the products of the heat- inactivated reverse transcription reaction to the PCR mix and proceeding with thermal cycling. As a general example, reaction volumes outlined in this procedure represent the addition of 5µl or 1µl fraction of the reverse transcription reaction to 25µl PCR amplifications. The volumes may be scaled for reactions less than 25µl. Carryover concentrations of magnesium chloride, PCR Nucleotide Mix, buffer and primers must be considered when combining the PCR mix components. 2. Prepare the PCR mix, minus the cDNA sample, by combining the components in a sterile, 1.5ml microcentrifuge tube on ice. Combine the components in the order listed, vortex gently to mix and keep on ice prior to dispensing to the reaction tubes. In this example, the final volume of PCR mix should be sufficient for a final reaction volume of 25µl once the cDNA volume is added. Scale the volumes to accommodate the total number of PCR amplifications being performed. Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Printed in USA. Part# TM316 Revised 12/12 Page 9 tm316.1212:EIVD_TM.qxd 12/18/2012 8:29 AM Page 9 Promega Corporation · 2800 Woods Hollow Road · Madison, WI 53711-5399 USA Toll Free in USA 800-356-9526 · Phone 608-274-4330 · Fax 608-277-2516 · www.promega.com Part# TM316 Printed in USA. Page 10 Revised 12/12 4.C. PCR Amplification for Endpoint Analysis (continued) Due to the ionic conditions, magnesium concentration a