qPCR data analysis - comparison of different genes in the same tissue

Question

[eszterhegyiova]

Hi all! I am facing a problem, I tried to perform qPCR analysis of three different genes from the same tissue (pancreas cDNA), and I would like to compare their expression. Can you help me, what method will be the best for this? I made absolute quantification with standard curves, but in this case I think I can only compare the expression of the same gene from different samples, but what I want to do is to compare the expression of three different genes in the same sample. Thank you all for your suggestions! eszter

[Devon Ryan]

Why don't you think you can directly compare absolutely quantified values between genes? You know something of the form "copies/µl".

[eszterhegyiova]

Yes, I did it. But I got strange results. I am using three primer pairs, one should amplify both genes (non specific, A+B= total), and two other are specific for each gene (A and B). But in my results A+B did not match the copy# I got when I amplified with the non specific primer. And there are huge differences. I thought, maybe the optimization of the reaction is not good, but the standard curves look good, the efficiency for the genes is around 105%, so it seems I can compare the results. Thank you!

[Devon Ryan]

This suggests, to me at least, that at least one of the primer pairs isn't specific enough. Are you using sybr green or taqman probes? Sybr green is obviously more prone to non-specificity issues.

[TriS]

You can compare them as long as your primers have the same high efficiency. In this case you can get the quantification of 2 relative to the third one. Otherwise what don't u simply run something like beta action and show the ratio to it?

Answer 1

[eszterhegyiova]

I am using SYBR Green. The 2 genes (A and B) has a similarity of 90% in their sequence, and it was really hard to design specific primers...