CT of qPCR - (Jul/03/2013 )

Hi everyone,

I’m fairly new to qPCR and I’m not sure how to troubleshoot the problem I have.

I’m running a duplex qPCR for two splice variants of my protein of interest. I’m using Syber since I’m more interested in the melting curve at the end, to screen for the existence of the isoforms. And that actually works. I can detect single peaks at different temperatures for homozygous or double peaks for heterozygous forms. My template is genomic DNA.

The problem is that I have to go up to 45 cycles to do this. As far as I understand qPCR should give a CT value of not to much higher than 25 to be trusted. My CT values are always between 33 and 36. I have increased primer, screened from 10 pmol and 50 pmol, increased template concentration, screened from 35 ng to 140 ng, added various amounts of Mg, tried different reaction volumes, 25 and 50 ul and used various annealing temperatures. Nothing seems to work. I always get the right melting curves but the CT does not change at all. Always 33 – 36.

Is there anything else I can try? Any suggestion is greatly appreciated.

C

I should also mention that I verified the result by running the qPCR product on a gel. I get the correct size fragments and no unspecific amplification.

Sounds a bit low if that is your primer concentrations. I use between 200 and 900 nM per primer!

You can also try and do a gradient PCR to optimise the annealing temperature, so you get better ampflication and earlier Cq values.

How much DNA are you adding?

 

What's wrong with a CT higher than 25? qPCR is a sensitive technique! Setting such a low threshold cripples the sensitivity of your assay.