qPCR with low expressing genes - (May/29/2011 )
I am doing qPCR experimetns using a delta delta ct analysis. Some of the genes I am looking at have been fine, but others have very low expression. At the moment I am making my standard curves using a dilution series made from pooled RNA samples. However, I cannot get more then 3 standards with this method with some of my genes. After three dilutions there is no amplification at all. I originally did 1 in 10 dultion series but I have reduced this down to 1 in 3 dilution series, but still can't get five points in the standard curve. I could use DNA standards instead of RNA standards but I have read that this is not ideal so would rather avoid DNA standards. I have also screened different tissues to see if any of them have higher expression of these genes, but no luck so far. Any suggestions for getting a standard curve with the genes?
I make only 3-point dilution, there is no rule that it has to be five, just that they have to cover the whole spectrum of your samples. I make 3 10x dilutions and run each dilution point in triplicate. So if you have only three or four dilution points that's IMHO sufficient.