ChIP-qPCR gives odd amplification plot
Posted 02 August 2011 - 09:50 AM
I was hoping for some help with a problem analyzing my ChIP-qPCR. My amplification curves are shaped oddly and sometimes sigmoidal. When I look at raw fluorescence, I see that it starts to come up above background in early cycles, levels off, and comes up again. When I run a gel on the product, I only see my intended amplicon.
I think it may have to do with my fragment size (200-600bp), but am not sure what I can do about it during analysis. I've been trying to fiddle with the baseline settings, but have not been terribly successful. Any advice would be appreciated!
Posted 02 August 2011 - 10:40 AM
Posted 02 August 2011 - 02:28 PM
you amplicons may be too big, mine are no more than 150 and most are ~75bp..........also, SYBR green wont discriminate between intial DNA and amplified DNA, so you may need to back off a bit in the DNA content. It could also be mispriming in the initial few rounds of PCR
Posted 02 August 2011 - 06:38 PM
Posted 08 August 2011 - 10:25 AM
Its just one of the two ChIP samples that gives sigmoidal curves on a small subset of primers.
The samples I'm using are actually amplified libraries made for ChIP-SEQ and were quantitated using the Qubit fluorescence reader. What I'm trying to accomplish here is validation of the sequencing results. I ran the primers on my input sample and genomic DNA as you suggested and it gives normal looking amplification curves.
I'm worried that my fragments are smaller than I think and my amplicons are too big for them. Would this cause the results I've seen?
I made all my primers with PerlPrimer using the same parameters. I designed them specific to binding sites and neighboring non-binding sites so that I could validate the sequencing results. I guess the biggest problem is that my qPCR data does not validate the ChIP-SEQ! The input shows enrichment of binding regions in some cases, but not in others.
Edited by mhong, 08 August 2011 - 12:33 PM.
Posted 09 August 2011 - 02:19 PM