9.3.4.1. Reverse transcription

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

1. Mix:
1.1.  0.5 μg       sample RNA template,  
1.2.  1 ng          exogenous reference RNA (e.g. Ambion RNA250),
1.3.  1 µl           50 ng/μl random hexamers,  
1.4.  1 µl           10mM dNTP,  
1.5.  up to 12 µl RNAse free water.
2. Heat the mixture to 65°C for 5 min and chill quickly on ice.
3. Add:
3.1.  4 μl 5X First-Strand Buffer,
3.2.  2 μl 0.1 M DTT,
3.3.  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 tenfold with nuclease-free water before using in PCR assays, to reduce the risk of PCR artefacts.