Two-Step RT-PCR Protocol

Place sterile, thin-walled dilution tubes and reaction tubes on ice. Thaw the experimental RNA or 1.2kb Kanamycin Positive Control RNA on ice, and return any unused portion to the freezer as soon as aliquots are taken.

On ice, combine the RNA (up to 1µg) and cDNA primer in Nuclease-Free Water for a final volume of 5µl per reaction.

Experimental Reactions

*We recommend 10² –10¹⁰ copies of a specific target RNA template or 1pg–1µg total RNA or poly(A)+ mRNA.

Positive Control Reaction

Negative (No Template)Control Reaction

Close each tube of RNA tightly. Place the 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.

Prepare the reverse transcription reaction mix by combining the following components of the ImProm-II™ Reverse Transcription System in a sterile 1.5ml microcentrifuge tube on ice. Determine the volume of each component needed for the desired number of reaction, and combine the components in the order listed. Vortex gently to mix, and keep on ice prior to dispensing into the reaction tubes.

Experimental Reactions

*The final Mg²⁺ concentration should be optimized in the range of 1.5–8.0mM.

Positive Control Reaction

Negative (No Reverse Transcriptase) Control Reaction

Dispense 15µl 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, giving a final reaction volume of 20µl per tube. If desired, overlay the reaction with a drop of nuclease-free mineral oil to prevent evaporation and condensation.

Anneal: Place the tubes in a controlled-temperature heat block equilibrated at 25°C, and incubate for 5 minutes.

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 37–55°C.

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.

Prepare the PCR mix by dispensing the appropriate volume of each component into 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. An aliquot of the first-strand cDNA (1µl or 20µl) from the reverse transcription reaction is added last to the PCR mix to give a final reaction volume of 100µl per tube. Overlay the reaction with two drops of nuclease-free mineral oil to prevent evaporation and condensation. See Notes 1–3. Use the Upstream Control Primer and Downstream Control Primer to amplify the 1.2kb Kanamycin Positive Control RNA.

*For experimental systems, the final Mg²⁺ concentration should be optimized in the range of 1.5–2.5mM.

Place the reactions in a thermal cycler that has been prewarmed to 94°C. An optimized program for amplification using the Upstream and Downstream Control Primers provided with this system is given in Table 1.

Table 1. Amplification Conditions for the Positive Control Reaction.

1. Denaturation: 94°C for 2 minutes
2. 25 cycles:
   • Denaturation: 94°C for 1 minute
   • Annealing: 60°C for 1 minute
   • Extension: 72°C for 2 minutes
3. Final extension: 72°C for 5 minutes
4. Hold 4°C

Information about RT-PCR optimization for other target RNAs can be found at: http://www.promega.com/paguide/chap1.htm.

After the cycle is complete, analyze the products or store the amplifications at –20°C.

Analyze the PCR products by agarose gel electrophoresis of 10% of the total reaction. The products will be readily visible by UV transillumination of an ethidium bromide-stained gel. The amplification product obtained using the Positive Control RNA with the Upstream and Downstream Control Primers is 323bp long.

Store the reaction products at –20°C until needed.

Notes

1. In this example, the final volume of PCR mix should be sufficient for 100µl reactions once the cDNA volume is added. The volume of each component may be scaled for reactions of less than 100µl. Scale up the volumes to accommodate the total number of PCR amplifications being performed.
2. The amount of reverse transcription reaction used in the PCR may be modified after experimental optimization.
3. Because of the ionic conditions, magnesium concentration and dNTP concentration of the reverse transcription reaction, the amount of magnesium and dNTP added to the PCR varies, depending on how much RT reaction is used as template. For example, for a 100µl PCR that contains 20µl of RT product, 8µl of 10X thermophilic polymerase reaction buffer is added to support the 80µl PCR mix addition. If 5µl of RT reaction is added to 95µl of PCR mix, 9.5µl of 10X thermophilic polymerase reaction buffer would be needed. Similar considerations must be given to the magnesium and dNTP additions.

Additional information about two-step RT-PCR using ImProm-II™ Reverse Transcriptase can be found in the ImProm-II™ Reverse Transcription System Technical Manual at: http://www.promega.com/tbs/tm236/tm236.html