How to compare different expression levels between genes - (Nov/25/2009 )
I'm wondering if it is possible to determine the "total" transcript levels of different genes in the same sample by standard qPCR, and if so what formula to use when calculating the values. I have already fairly nice standard curves based on 5-step dilutions, and the primer efficiency varies between 0,95 and 1,05. I presume that it's not enough to do the delta delta C(t) and substitute the 2 with 1 + primer efficiency? And can you disregard that a longer amplicon will yield a higher signal than a shorter one, or does that also have to be considered. This might be simple question, but i haven't found a proper answer in the literature (although i must admit that the search was quite brief).
-Christoffer-
Christoffer on Nov 25 2009, 10:42 PM said:
I'm wondering if it is possible to determine the "total" transcript levels of different genes in the same sample by standard qPCR, and if so what formula to use when calculating the values. I have already fairly nice standard curves based on 5-step dilutions, and the primer efficiency varies between 0,95 and 1,05. I presume that it's not enough to do the delta delta C(t) and substitute the 2 with 1 + primer efficiency? And can you disregard that a longer amplicon will yield a higher signal than a shorter one, or does that also have to be considered. This might be simple question, but i haven't found a proper answer in the literature (although i must admit that the search was quite brief).
Since you have the standard curves, use the Pfaffl method. You can google Pfaffl, get the 2001 paper and read. It is more accurate than delta delta Ct. You can also read this, a good beginner's guide to realtime PCR:
http://pathmicro.med.sc.edu/pcr/realtime-home.htm
Chris
-chrisbelle-