Is one reference gene ever appropriate? - (Jun/15/2011 )
So I am relatively new to qPCR and have read much in the literature about not using single reference genes and trying to use as many as three different genes for normalisation. However, if the Ct values are identical between runs for a reference gene, does this not show its stability? In this case, is one gene OK, or are more still needed?
I am also trying to get the answer of this question? If you find please intimate me.
I would say that one stable housekeeping gene is better that three unstable ones. If your gene is stable, then it should suffice, but if you're writing a paper, reviewer may require more. Or not, that depends. Some people publish suspicious results with actin for years because "it's used everywhere" not caring about the real stability in certain situation. So if you say you tested five housekeepings and selected two most stable ones, it sounds better. Maybe you can test more and use two best, or just write, that you considered the stability and this one was stable so you use it in normalisation.
It is generally recommended to work with several reference genes, ideally three or more. I agree with the previous post that one stable reference is better than three unstable ones... but what about using three stable ones?
There's a free online tool called RefGenes that helps you to find candidate reference genes for tissues of interest. The tool is open access and available at www.refgenes.org. RefGenes compiles data from thousands of microarrays and gives you the most stable genes for a chosen set on microarrays (i.e. those done on your tissue of interest).
- Philip, Zurich, Switzerland