Protocol Online logo
Top : New Forum Archives (2009-): : ChIP and Next Generation Sequencing

ChIP data normalization - (Dec/31/2011 )

Hi, I am wondering how best to normalize my ChIP-qPCR data.

I did H3K9me3-ChIP on samples from two different tissues.to compare the %Input at my gene of interest. One of the tissues showed reproducibly higher levels of H3K9me3 than the other tissue (n=4 independent replicates), but the interesting thing is that the positive control primer in the heterochromatin also gave consistently higher H3K9me3.

Now, the question is whether the ChIP values are indeed higher in both the gene of interest and the positive control primer (true positive), or whether the consistently higher values are caused by a systematic error in sample handling (false positive). I am thinking whether I should normalize my %Input with respect to the positive control primer to make the positive control score constant, and then read the new %Input at my gene of interest.

Do your positive control primers always have very similar values across biological replicates?

Or do people recommend any other methods of normalization?

-Serendipitous-

I would recommend normalizing your data to a negative control regions % Input

-chabraha-

I agree with chabraha...I've mentioned before around here, I like LINE1 as a good negative control region because its transcription is actively repressed, but there are lots of copies of it in the genome, so it comes up nice and clean in your qPCRs, whereas if you use an intergenic region you might get a lot of variability because you may not pull down a lot of it...

-Mighty Mouse-