11.8. cDNA synthesis and template quantification
first-strand reactions consist of a 20µ volume containing:
0.5-2 mg of total RNA, or 50-100 ng of poly(A)RNA,
pmol of anchored d(T)20VN primer,
200 units of Superscript III (Invitrogen),
1X concentration of proscribed buffer.
- The tube is incubated for 1 h at 50°C.
- Terminate by adding 30µl of TE buffer and freezing.
- Resulting cDNAs can be screened for levels of specific genes via
quantitative-PCR as described in the RNA methods in section 4.7.