I had one question concerning comparison of samples for the purpose of qRTPCR. So, I made all my cDNA samples (treated vs untreated) with 1ug of total RNA in 20ul of volume. However, for one of my samples, I was low on RNA, so I made cDNA from 500ng of RNA in 10ul of volume so that concentration of cDNA for comparison is same. Is this a valid way to get Ct value comparison?
Not ideal, but I think that it should be ok so long as you are comparing the expression of each gene of interest to an internal reference gene (ie. actin or tubulin). Compare the individual biological replicate Cts to one another and see if there are any major differences (ie. whether your smaller cDNA prep may have introduced some sort of bias).