I have a couple of questions about designing experiments to check my reference genes. In my lab they're not very concerned with this but having read the literature I think it is important. In my previous lab I did this using Taqman assay and relative quantification. Now, in this lab, we use SYBR green assays and the absolute quantification method. Is testing for the most stable reference gene the same with absolute quantification as with relative quantification?
My specific questions are:
1) For the standards we use plasmids, but having read some posts on this forum I'm not sure this is the best approach since it doesn't compensate for the efficiency of reverse transcription. Is there a better option for the standards? What are the drawbacks of this better option? How would one measure the RT efficiency and is there a way to incorporate this into the quantification?
2) I know what is required for each quantification method (standards, ref genes, calibrator...) but from everything I've read the following is still unclear to me: is it possible to do absolute quantification with the Taqman assay and relative quanitifcation with the SYBR green assay, and vice versa?
3) Do I have to run a reference gene on every plate? Or is it ok if, for example, the ref gene is on plate number 1 and I compare target genes on plates number 1-5 based on this reference gene?
4) I have RNA samples from 3 different conditions, 2 wells per condition (so this would be the biological replicate, yes?), in 3 experiments (n=3). Can I run all the samples from the 3 experiments on one plate, or does it have to be 3 different plates?
5) About Normfinder, geNorm and Bestkeeper - do I enter raw data, so just Ct values, or does it have to be quantified? Is it possible to use these programs with absolute quantification?
6) For my pilot experiment where I measure the stability of reference genes across samples and experimental conditions I had 3 genes - b-actin, g-tubulin and b2-microglobulin, I had standards from plasmids for each of the three, and I had samples for experiment no.1. I averaged the Ct values of the triplicates, and then averaged the biological replicates. Is this ok? Is the difference in Ct value alone enough to tell me which gene is the most stable - where the gene with the least variable Ct value between all samples would be the most stable gene. Do I need to run this with samples from the other experiments?
Thanks! I know its a long post, but I'm swimming in this sea of qPCR literature all alone and any life-raft would be greatly appreciated!