I'm quite new to the RT-PCR field, and for my internship I have to do a couple of RT-PCR experiments using SYBR green. I've read a couple of documents about it and it seems that validating the primers is very important however, it is not totally clear to me yet.
For instance: Since I have multiple knockdowns of a cell type, should I make a standard curve of each of those knockdowns? Or is testing the primers on the Wildtype cell line enough?
Also, should the forward and reverse primer be tested seperately or in the same well?
Concerning the plate lay-out: I was thinking of doing the standard curves in triplicates (5 log dilutions) and taking along a H2O sample for each primer to test for primer dimers. Is that enough or should I take other controls into account?
Thanks in advance!
Edited by Aloys86, 12 July 2010 - 05:33 AM.