Reverse transcription 

The goal of this step will be to perform the reverse transcription of mRNAs into CDNAs. To this avail, we will employ an Oligod(T) primer that binds to the poly(A) tail of the mRNAs. Therefore, only mature poly(A)-tailed mRNA will be reverse transcribed (not rRNAs, microRNAs, etc).

Material

  • Thermocycler
  • PCR tubes
  • Oligod(T) primers
  • dNTPs mix 10 mM
  • MMLV buffer 5X (= Reverse-transcriptase buffer)
  • MMLV enzyme (200 U/µl) = Reverse transcriptase enzyme, one stock for all groups. A detailed protocol can be found at https://tinyurl.com/yvfdexf9
  • RNAse inhibitor (=RNAsin 20 U/µl ; one stock for all groups)
  • Sterile water

Procedure

In this procedure, we will first need to calculate the volume of samples to use based on the quantification of the RNA that was performed at the previous step. Ideally, we will perform the RT on 1500 ng of RNA.

  1. Calculate the volume of samples 1 and 2 to take to reach a total of 1500 ng of RNA in a maximum of 11 µl. If this is not possible because your yields were lower than 136 ng/µl, calculate the amount of RNA contained in 11 µl of the sample with the lower yield (e.g. 87 ng/µl = 957 ng total RNA in 11 µl). For the other sample, calculate the volume that you need to reach to have the same total amount of RNA.
  2. Pipet these volumes and add them to previously-annotated new PCR tubes.
  3. Add 1 µl of oligod(T) (0,5µg/µl) to each of the samples.
  4. Add water to a total volume of 12 µl (formula = 12 µl - Vol samples - 1 µl)
  5. Centrifuge and incubate for 5 min at 65°C in a thermocycler.
  6. Chill on ice.
  7. Add the following components in that order to each tube:
    • 4 µl of MMLV reaction buffer 5X.
    • 1 µl RNase Inhibitor (20 U/μl) = RNAsin.
    • 2 µl dNTP Mix 10 mM.
    • 1 µl MMLV Reverse transcriptase (200 U/µl).
  8. Incubate for 60 minutes at 42°C (thermocycler) for the reverse transcription.
  9. Terminate the process by heating at 70°C for 5 minutes.
  10. Add 50 µl of sterile water to a final volume of 70 µl.
  11. Mix by up-and-down pipetting and proceed to the qPCR part of the protocol.

Note:

Because of the cost of the reverse transcription (the most expensive part of the protocol, due to the cost of the reagents), we will not perform a RT-control reaction for each group. However, we usually perform such a control by adding all the reagents (oligod(T) + MMLV Buffer + MMLV + dNTPs + Water) without any RNA sample. When performing the RT-qPCR, this sample can help us determine whether the reagents used for the RT were contaminated (in case an amplification occurs in this sample, it suggests that reagents were contaminated).