ChIP-CHIP IP vs IgG vs Unbound vs Input - (Feb/24/2009 )

Hi all,

Was wondering if anyone could provide any insight as to why IP is compared to input in ChIP-chip and qPCR experiments? Any reason why I can't compare to the non-captured (unbound) fraction and hybridize to an array? Seems like doing it this way will result in larger differences between IP and Unbound fractions at regions which are enriched by the antibody?

To give you an idea we're getting ~4-5 fold enrichment at our positive control genes compared to input, but about 50 - 60 fold enrichment compared to negative control genes which should not be enriched. When we compared to unbound, we're getting about 50 - 300 fold enrichment at our positive control genes, while maintaining depletion of our negative control genes. Any reason why comparing to INPUT or UNBOUND wouldnt work.

Any thoughts would be helpful.

Thanks in advance,

Steve

Steve,
I apologize to just piggy back onto your post, but I could not, for the life of me figure out how to post something...

I have a question about how to analyze the ChIP data that I have. I have looked through several posts and lots of people seem to have the same question I do, but I have yet to see an actual answer. I am very new to this whole technique, so my questions will be very basic.

I want to eventually do ChIP-Chip, but first need to verify that the IP actually worked. I have run RT-PCR using SYBR green. For the input sample I diluted it so that for the IP and the input I put in not only the same volume, but the same amount of DNA. The negative control is obviously the problem, as most people have seemed to say. This sample is obviously more dilute than the others, so I simply used the same volume for this sample as well, but this would obviously have much less DNA. I am not sure if this is how I should set this up, but that's the best I could come up with. I have CN and Ct numbers now, but have no idea what to do from this point. I am supposed to figure out if the IP 'enriched' for the gene of interest, but am not sure how to go about that. Oh, and just for further information, as of right now, I am using an H3 antibody (pan), which should bind to all DNA sequences. I moving towards histone modifications, but need to know how to analyze this data first. I would appreciate anyone who can help me with this, as I have exhausted all avenues that I can find.

Thanks again.... and sorry Steve....

Hi Steve,

We do loads of ChIP-chip and always hybridize our IPs with Input. Why? Well, we don't want to compare our IPs to crap do we? (ie: non-specific binding). If you wanted to be really anal in your experiment you would hyb IP vs IgG and IP vs Input for each sample, but who has enough money to do that?

Clare

emacalate on Feb 25 2009, 03:14 AM said: