Relative quantification of different genes in single sample - same threshold val - (Dec/07/2011 )
Hi, I am doing gene expression study using real time PCR. The machine model is ABI Prism 7000 Sequence Detection System.
I would like to know, by using real time relative quantification application to compare the gene expression level of few gene isoforms in a single cancer cell line, do I need to use same threshold setting (ex. Manually set threshold at 0.2) for all primers or just choose ‘auto’ threshold setting for each primer gene before collecting each Ct value?
Besides that, I would like to ask is that appropriate to use comparative Ct method <2–(∆∆Ct)> to compare the gene expression of different isoforms in a single cancer cell line if I would like to use relative quantification method? Or any recommended formula to be used?
Someone will tell me if I am mistaken, but.....delta-delta-CT method can only tell you the relative levels of a single gene across multiple samples, not the relative quantities of different genes or isoforms. For your purposes, you will need to generate a standard curve where you know the absolute quantity of the samples within the standard curve so that you can calculate the quantity of each mRNA in your one sample.