Inconsistent duplos - (Jan/06/2006 )

Hi,

I have recently started using ChIP, and I have some problems with the quantification.

To check for enrichment, I perform Taqman PCR of immunoprecipitated chromatin in duplicate reactions. However, the Ct-values of my duplos often differ by more than 1 Ct. This is especially the case for my negative controls (IP with rabbit IgG or no Ab), but also IP-samples and pos controls (anti-H3K9), whereas the Ct-values of input chromatin are very comparable between duplos (often less than 0.1 Ct difference) .

Also when I just use cDNA for my PCR I have almost always less than 0.1 Ct difference between duplos, so it is not the pipetting.

Did anybody have the same problem and found a solution?

The fact that your input DNA and your cDNA give very consistent results but your ChIPed DNA doesn't suggests that the problem is with low concentration of your ChIPed DNA. Are your Ct for the ChIPed DNA at the high end? There may be a couple of fixes for this. If you are diluting your ChIPed DNA before running it try running it without diluting. In the future your best bet is to start with a higher input concentration of DNA for your ChIP, which basically means more cells. I usually use a little less than two 10cm plates (I never count the cells but they're a primary fibroblast like cell at about >80% confluence) per sample. With this number of cells my triplicates on real time with cyber green typically have a std dev of less than 0.1 for my IPs and 0.1 to 0.3 for my mock IPs (if you're not doing mock IPs you could probably decrease the number of cells).

I was having the same problem as you but it went away as soon as I started using more cells.

Good luck,
Joel