Maximum acceptable variation of Housekeeping gene - (Jun/15/2007 )
I have a relatively simple question which may not have a simple answer ( I have been browsing the net for answers, but there is no concensus to find).
Does anyone know, or have a general idea what generally is considered to be an acceptable difference between expression of a housekeeping gene of sample vs control (for instance treated vs untreated, or cancerous tissue vs noncancerous tissue)?
Below 0.5 Ct, between 0-1 Ct or maybe even above that?
Or is it maybe a percentage value? For instance, if the difference between treated and untreated sample of the target gene has an average value of 3 Ct, then the acceptable difference between treated and untreated sample of the housekeeping gene should be smaller (let's say 0.5 Ct) than if the difference between treated and untreated sample of the target gene has an average value of 6 Ct (let's say 1 Ct). In other words, a shift of housekeeping gene expression of 16.67 %.
I am fully aware that no housekeeping gene is ideal.
Personally I prefer using the Pfaffl method for calculating gene expression (Pfaffl,M.W. (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res., 29, 2002–2007.)
Thx in advance
As far i have reading the acceptabçle difference for HSG is something like 0.5. Both methods, Pfaffl and the deltaCt, take in acount the HSG variation, using a diferent way to do it. In my case does not always happen that my HSG is invariable... that's bad. Anyway, concerning what i have been reading and what people told me the 0,5Ct is the maximun.. the same for the replications.