Negative Control Primers for ChIP Assay
Posted 15 July 2009 - 08:59 AM
Posted 15 July 2009 - 10:03 AM
Hi! I have a problem with getting a right negative control for my Chip experiment. I am working with a mouse immortalized cell line and was very happy to see the recruitment of the TF of my interest at the expected binding site. But the bad part is that where ever I look for it i see enrichment over IgG ( i have checked 3-4 supposedly negative primer sets). I have looked upto 15000 bp away from the proximal promoter and even inside the gene. Does this make any sense? If this is a false positive then what could be the reasons. I know that the antibody i am using works very well. Any suggestions? Are there any bonafide negative controls for Chip experiments. I am new to this technique.
The difficulty with looking at enrichment over mock IP is that, even if you use non-immune IgG as a mock, there are still differences between the mock and specific sera. Which means you can see a difference in non-specific binding between the two which does not represent specific binding. For instance, when I used Abcam's H3K4m3 antibody in ChIP I would typically get an enrichment over mock of about 50-fold at my negative control region. When I looked at the TSS of a well expressed gene, however, I saw a 500-1000 fold enrichment. So I don't consider the enrichment over mock to be of much value. The one thing that matters is that you can show a difference between your negative control region and your region of interest.
Posted 14 December 2010 - 09:25 AM
I have had similar problems with my assay. My current study involves trying to locate where on our target gene a transcription factor of interest binds. I have been ChIPing using three different antibodies all directing against my TF of interest. I also include a positive control antibody and a negative control antibody. Following immunoprecipitation and DNA purification I have been running qRT-PCR to amplify regions of a gene where we believe our TF might bind. I have been getting a signal relative to my negative control antibody for two out of the three target antibodies (and my positive control). I am not sure why the one antibody does not give me a signal greater than the noise. Nevertheless, the real problem that I have been having is trying to understand my qRT-PCR results. When I try different primers for different areas of the gene I get no change in the rank order of signal to noise between my samples. Although the signal to noise changes, the difference between the target antibodies and the positive control remains constant. Recently, to try and make sense of this, I used the same DNA and ran a PCR with primers of a gene that is not even expressed in our cell line and got similar results as before. I am trying to ponder how I can get amplification of my DNA using primers that are directed to a gene that is not even expressed in my cell line! If anyone can shed light on this dilemma I would greatly appreciate it. Thank you.
Posted 29 December 2010 - 11:25 AM