11.8. cDNA synthesis and template quantification

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