How do we calculate Ct values for candidate reference genes?
I’m trying to determine an appropriate reference gene for my real-time experiments (treated vs untreated) and I’m a little confused as to the determination of Ct values for my candidate reference genes. To my knowledge, a standard curve is required and this is where my confusion lies.
Without a standard curve, I am unable to determine both the Ct values and relative copy number for my genes and without either of these, I can’t compare expression variation. So, really, how do I know which gene to use to generate my standard curve?
I’ve imported standard curves from other experiments (efficiency > 0.99) and the differences in the Ct range from 0.3 to 0.5. This then leads to a copy number difference of up to 20 million copies!
Am I even asking the right question/approaching this correctly?