Reverse-transcription
To remove contaminating DNA, It is recommended that RNA be treated with DNase using the DNA Free kit (Ambion). This involves incubating 10 mg of RNA at 37°C in a 50 ml volume reaction, containin of 2U rDNaseI, 5 m l 10x DNase I buffer for 30 minutes. Following incubation 5 ml of DNase inactivation reagent is added to the RNA, and mixed by pipette. Following a 90 second spin at 10000 x g in a microcentrifuge, the supernatant, which contains the RNA is transferred to a fresh Eppendorf.
Construction of cDNA using reverse transcriptase
Superscript II Reverse transcriptase (Invitrogen) is used to construct cDNA. ~2 mg of DNA free treated RNA is mixed with 1 ml Oligo d(T) in an Eppendorf tube, and incubated at 70°C for 10 minutes. The Eppendorf tube is then transferred to ice and the following components added, 4 ml 1st strand synthesis buffer, 2 ml 0.1M DTT, 1 ml 10mM dNTPs, 1 ml RNase inhibitor, 1 ml Superscript II reverse transcriptase. The reaction is made up to 20 ml in nuclease free H20. The mixture is then incubated at 42°C for 45 minutes to allow the reverse transcription reaction to occur. The tube is then transferred to a 75°C water bath for 15 minutes, to destroy the enzymes. The tube is span down, and further diluted with the addition of 80 ml of nuclease free water. 10 ml of the product is used for subsequent PCR reactions.