Relative Quantification in real-time RT-PCR - (Jul/04/2006 )
Which method you recommend to use for relative quantification in real-time RT-PCR? delta-delta Ct, Pfaffl, or ...?
I prefer the standard curve method. It allows to normalize to another RNA (or DNA). And I calculate everything as on the Applied Biosystem's handbook
I like ddCt. I began with standard curve, then decided to switch to ddCt because of the number of genes we survey (the plates were very cumbersome and expensive)
I ran the two methods on the same samples in parallel for awhile (using variable conditions) to validate the method switch. Results were quite similar (as they should be ) so I switched entirely. Now I can squeeze more data out of a single plate and I can set up fewer samples.
Again, I'm for the delta delta Ct method.
I like the efficiency one. Get some RNA from the cell lines or tissues and try to get the efficiencies of each gene you are going to assay. You can read more at
delta delta method assumes that target gene and control gene have the same efficiency, which is 2 as predicted.
I´m using the ddCt, but now i´ve a problem, Can you help me? I use 3 samples for the same pacient (the first one (time zero), normalize the other two) but in my last samples the normalization samples are very bad(higher Ct values). Like this i´ve bad results because the gene expression is low. Is ther another way to get a relation of the gene expression?