relative quantification, primer pair problem - (Jun/15/2006 )
I have the following problem: I performed real-time with SybrGreen with two different primer pairs (designed with primer express, I call them A1 and A2) against the same transcript. These primer pairs are supposed to be common for three splice variants (A,B,C). One experiment with treatment was performed: A1, B and C are downregulated (relative quantification), but because A1 is the common part for all variants there should be something like A1=A1+B+C (is not true even for a absolute quantification). Same experiment: A2 is down, B+C are up. Does anyone have a idea about this problem?
I am not sure the reason for your problem. for relative quantification, first you should validate your primers. Applied biosystems USER2 Manual has the protocol for it. The efficiency of primers (to the same target gene) is different for many reasons. For this you have to carry out serial dilution (usually 10 fold) of cDNA and test the primers at this concentrations and compare them to your internal control. I usually select primers which are sensitive (they pick up signal early, pick up signal at very low concentration efficiently, and also, importantly, the difference between the Ct of test primer and internal control is same at various dilutions). Hope you are doing this way.