Reference genes on same plate as gene of interest? - (Aug/28/2006 )
I want to use 3 reference genes to calculate normalisation factors for my experiment. I know that ideally I should run the 3 reference genes on the same plate as my gene of interest but unfortunately this would require that I run my samples over many plates. Would it be acceptable therefore to run each reference gene on a separate plate?
If you're calculating gene expression based on absolute quantitation (ie. have a standard curve with every primer pair on every plate), then placing your reference genes on separate plates might be acceptable.
However, if you are planning to do delta-delta-Ct type analysis, then I would certainly avoid having the reference genes be located on a separate plate. If the ref genes aren't on the same plate as the target genes, then there is no way to control for differences in PCR efficiency from run to run, etc, and you could wind up with very misleading and/or erroneous results.
Thank you for the reply. I was wondering whether if it would be possible to have some kind of calibrator on each plate that would allow me to correct for interplate variation? Maybe I could make up a large batch of PCR components and freeze in aliquots for each plate?