how to evaluate chIP enrichment efficency? - (Aug/27/2006 )
Hi,
I am doing chIP-chip experiment to study a transcription factor X's targetting seqences.
right now I am doing chIP experiment from different cell lines. the first problem I have to solve is to make my chromatin IP work which means I can get enriched target DNA sequence when I performed a qPCR to evaluate the enrichment of my chIP experiment.
I just pick up a gene A that is considerd to be regulated by X, I design the primers to amplify A's promoter region which is supposed to be the targetting site of transcription factor X, and also design the primer which amplify a sequence that should not be target by X (this serves as negative control region), then I use realtime PCR to evaluate the enrichment of promoter region of gene A in chIP generated DNA compared to that in input genomic DNA. the results come out, they are not very exciting, some of cell lines did not have the enrichment, some of them have 2-3 fold enrichment, and also for a certain cell line, some time the result is 2-3 fold enriched, some time there is no enrichment at all. (why does this happen, different growth condition of cells when perform cross linking?)
So, I would like to know if you guys have the same situation as this, or I think I have to change another target to evaluate if my chIP work. but what is the best way or method to claim if the chromatin immunoprecipitation really works?
Thanks.
-Q
I am doing chIP-chip experiment to study a transcription factor X's targetting seqences.
right now I am doing chIP experiment from different cell lines. the first problem I have to solve is to make my chromatin IP work which means I can get enriched target DNA sequence when I performed a qPCR to evaluate the enrichment of my chIP experiment.
I just pick up a gene A that is considerd to be regulated by X, I design the primers to amplify A's promoter region which is supposed to be the targetting site of transcription factor X, and also design the primer which amplify a sequence that should not be target by X (this serves as negative control region), then I use realtime PCR to evaluate the enrichment of promoter region of gene A in chIP generated DNA compared to that in input genomic DNA. the results come out, they are not very exciting, some of cell lines did not have the enrichment, some of them have 2-3 fold enrichment, and also for a certain cell line, some time the result is 2-3 fold enriched, some time there is no enrichment at all. (why does this happen, different growth condition of cells when perform cross linking?)
So, I would like to know if you guys have the same situation as this, or I think I have to change another target to evaluate if my chIP work. but what is the best way or method to claim if the chromatin immunoprecipitation really works?
Thanks.
-Q
One possibility is that your TF is loosely associated with the DNA and may be bound more tightly in some cell lines then others due to the degree of complexing with other proteins at the promoter. To increase the efficiency of pull down of your TF you might try more crosslinking (the strongest conditions we use are 1.4% formaldehyde for 15min) or you may also try different crosslinking reagents or protocols. For instance paraformaldehyde can provide longer crosslinks than formaldehdye thus allowing for crosslinking of proteins more loosely associated with DNA. In addition, a two step crosslinking protocol has been used for pulling down NF-Kb where proteins are first crosslinked to each other to stablize protein complexes before protein DNA crosslinks are made (Biotechniques 2005. 39(5) p 715-725).