I have found that if you are using degenerate primers for RT-PCR,
you must optimize the annealing temperature, in my case, we started very
low (40 C) and worked up until we got a strong band using a positive
control mRNA. You could probably also add more template, but most of the
RT kits suggest an upper limit of 1ug RNA.
Other advice is running more cycles, 35 to 40, or taking the
product, and using some of that for a second round of amplification.
Good Luck,
French Lewis
