qPCR stats problem - Vandesompele's, DDct or other methods?? (May/13/2010 )
I have a qPCR statistics problem. Basically right now I am comparing gene expression between two groups of animals using only one house-keeping gene as my reference gene. Can anyone advise which method of analysis is the best? Some people have recommended Vandesompele's method of geometric averaging of multiple internal control genes, but I have difficulty understanding where the formula arises from and quite frankly it doesn't make much sense to me (especially given that I only have one reference!). Another method which I have been considering is just using the DDct method, and take the DDct of each animal to create my standard dev and a p value. Is that method acceptable or can anyone advise on any other methods?
Thanks in advance!
You said you have only one housekeeping gene, so why bother with averaging, if you think it's a stable gene, just use it as reference.
DDCt method doesn't acount for different efficiencies of the reference and target genes, so better way to quantify is to use efficiency corrected Pfaffl equation. You would need to run a dilution series to estimate the efficiency of each gene.