I've been working with the SN48 as my housekeeper for my RT-qPCRs, this seems like quite a stable housekeeper as it remains around 22-24 CT's consistently throughout the runs across different samples. Is a CT difference of 2 an acceptable max. for housekeepers?
I would suggest using a second housekeeper to be sure, if its values show the same differences then it should be fine.
Hi,
in my eyes the assumption that the housekeeping gene (HKG) is stable because the Ct value doesn't change is wrong. If this would be so, there would be no need for normalization, because you could normalize everyhting to the amount of cDNA. The problem with qPCR is that you don't know the amount of cDNA you use for your qPCR. You know the RNA concentration you use for cDNA synthesis, but only ~1% of this is mRNA and only a part of this will be reverse transcribed to cDNA. Measuring the absoption after cDNA synthesis won't give you the cDNA concentration as the solution still contains RNA and nucleotides which will also absorb at the same wavelength. There are several methods to find good HKGs. Take a look at GeNorm (Vandesompele et al. (2001) Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biology 3(7).) or NormFinder.
Hope this helps.
It would be better to use at least two house keeping genes. for detail see this paper http://clinchem.aaccjnls.org/content/55/4/611
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