4.5.1 Reverse Transcription of RNA

The following is a robust reverse-transcription protocol for generating cDNA that is fully representative of the original RNA population:

  1. Mix:
    0.5 μg         sample RNA template,
    1 ng            exogenous reference RNA (e.g. Ambion RNA250),
    1 µl             50 ng/μl random hexamers,
    1 µl             10mM dNTP,
    up to 12 µl  RNAse free water.
  2. Heat the mixture to 65°C for 5 min and chill quickly on ice.
  3. Add:
    4 µl 5X First-Strand Buffer,
    2 µl 0.1 M DTT,
    1 µl (200 units) of M-MLV RT.
  4. Mix by pipetting gently up and down.
  5. Centrifuge briefly to collect the contents at the bottom of the tube.
  6. Incubate 10 min at 25°C.
  7. Incubate 50 min at 37°C.
  8. Inactivate the reaction by heating 15 min at 70°C.
  9. Dilute the cDNA solution ten-fold with nuclease-free water before using in PCR assays.

 

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