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:
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.
- Heat the mixture to 65°C for 5 min and chill quickly on ice.
4 µl 5X First-Strand Buffer,
2 µl 0.1 M DTT,
1 µl (200 units) of M-MLV RT.
- Mix by pipetting gently up and down.
- Centrifuge briefly to collect the contents at the bottom of the tube.
- Incubate 10 min at 25°C.
- Incubate 50 min at 37°C.
- Inactivate the reaction by heating 15 min at 70°C.
- Dilute the cDNA solution ten-fold with nuclease-free water before using in PCR assays.