I have run qpcr on two different genes from the same family and I want to compare their levels to each other. I have normalized their expression values using GAPDH and eEF1e1 based on geNorm.
The problem I am having is whether or not I can arbitrarily set one time point from one gene to be 1 and then use the same factor to adjust the other gene? Does that make any sense?
Ex: The normalized 2CT value for gene A is 2.71x10^-11 and for the same time point gene B's normalized 2CT value is 1.35x10^-8. Can I divide them both by 2.71x10^-11 and say that gene B is expressed ~500x more than gene A? (Note: Gene B is well documented as being highly expressed and gene A is well documented as being very lowly expressed, so 500x more is expected)
Do I need to calculate in the efficiency of the PCRs to account for the fact that different products are being amplified? If so, how do I do that? Is there a paper I can read to help me out?
Thank you SO MUCH!!