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CHIP: No enrichment in IP DNA Relative to Input - (Mar/07/2007 )

I have been using the EZ ChIP kit from upstate and I have some technical questions about the ChIP protocol. Through small scale ChIP analysis and quantitative/conventional PCR I am able to see enrichment of RNA polymerase II bound DNA relative to the IgG control but I do not see enrichment of RNA polymerase II bound DNA relative to Input DNA (sonicated but not immunoprecipitated). Does anyone have any ideas how I can solve this potential problem? Traditionally people have assumed that enrichment of IP'ed DNA relative to non-immune control DNA is sufficient, but in reality the IP'ed DNA should be enriched relative to the Input. Any suggestions are welcome!

-kiddserono-

QUOTE (kiddserono @ Mar 7 2007, 06:53 AM)
I have been using the EZ ChIP kit from upstate and I have some technical questions about the ChIP protocol. Through small scale ChIP analysis and quantitative/conventional PCR I am able to see enrichment of RNA polymerase II bound DNA relative to the IgG control but I do not see enrichment of RNA polymerase II bound DNA relative to Input DNA (sonicated but not immunoprecipitated). Does anyone have any ideas how I can solve this potential problem? Traditionally people have assumed that enrichment of IP'ed DNA relative to non-immune control DNA is sufficient, but in reality the IP'ed DNA should be enriched relative to the Input. Any suggestions are welcome!


When you say that you don't see any enrichment vs. input DNA do you mean that you dilute your input DNA down to the same concentration of DNA in your IPs before running PCR and then don't see any difference in signal at a site likely to be bound by Pol II (active gene). Or do you mean that you don't see any difference between input and IP'd DNA in the enrichment of a site likely to be bound by Pol II over a site not likely to be bound to Pol II (silent gene)

-KPDE-

Youre assuming that IP efficiency is 100% and it is not... Probably way lower than 100%

-beccaf22-

QUOTE (KPDE @ Mar 7 2007, 03:00 PM)
QUOTE (kiddserono @ Mar 7 2007, 06:53 AM)
I have been using the EZ ChIP kit from upstate and I have some technical questions about the ChIP protocol. Through small scale ChIP analysis and quantitative/conventional PCR I am able to see enrichment of RNA polymerase II bound DNA relative to the IgG control but I do not see enrichment of RNA polymerase II bound DNA relative to Input DNA (sonicated but not immunoprecipitated). Does anyone have any ideas how I can solve this potential problem? Traditionally people have assumed that enrichment of IP'ed DNA relative to non-immune control DNA is sufficient, but in reality the IP'ed DNA should be enriched relative to the Input. Any suggestions are welcome!


When you say that you don't see any enrichment vs. input DNA do you mean that you dilute your input DNA down to the same concentration of DNA in your IPs before running PCR and then don't see any difference in signal at a site likely to be bound by Pol II (active gene). Or do you mean that you don't see any difference between input and IP'd DNA in the enrichment of a site likely to be bound by Pol II over a site not likely to be bound to Pol II (silent gene)


When I finish the CHIP protocol and nanospec the DNA I see basically the same concentration of Pol II and IgG IP'ed DNA. Then in performing PCR I use the same amount of DNA (also the same volume). My goal is to do CHIP CHIP so I need something to normalize to. I think using the input to normalize would be better than using the IgG control. For example, if "protein X" binds 1000 promoters I should see enrichment of these sequences in protein X IP'ed DNA relative to Input or IgG control DNA. Even though the IP'ed DNA may have less overall DNA, the IP'ed fraction should be enriched for protein bound sequences relative to the Input. So my question is what suggestions do you have to see better enrichment in the IP'ed fraction?

-kiddserono-

QUOTE (kiddserono @ Mar 7 2007, 02:12 PM)
QUOTE (KPDE @ Mar 7 2007, 03:00 PM)
QUOTE (kiddserono @ Mar 7 2007, 06:53 AM)
I have been using the EZ ChIP kit from upstate and I have some technical questions about the ChIP protocol. Through small scale ChIP analysis and quantitative/conventional PCR I am able to see enrichment of RNA polymerase II bound DNA relative to the IgG control but I do not see enrichment of RNA polymerase II bound DNA relative to Input DNA (sonicated but not immunoprecipitated). Does anyone have any ideas how I can solve this potential problem? Traditionally people have assumed that enrichment of IP'ed DNA relative to non-immune control DNA is sufficient, but in reality the IP'ed DNA should be enriched relative to the Input. Any suggestions are welcome!


When you say that you don't see any enrichment vs. input DNA do you mean that you dilute your input DNA down to the same concentration of DNA in your IPs before running PCR and then don't see any difference in signal at a site likely to be bound by Pol II (active gene). Or do you mean that you don't see any difference between input and IP'd DNA in the enrichment of a site likely to be bound by Pol II over a site not likely to be bound to Pol II (silent gene)


When I finish the CHIP protocol and nanospec the DNA I see basically the same concentration of Pol II and IgG IP'ed DNA. Then in performing PCR I use the same amount of DNA (also the same volume). My goal is to do CHIP CHIP so I need something to normalize to. I think using the input to normalize would be better than using the IgG control. For example, if "protein X" binds 1000 promoters I should see enrichment of these sequences in protein X IP'ed DNA relative to Input or IgG control DNA. Even though the IP'ed DNA may have less overall DNA, the IP'ed fraction should be enriched for protein bound sequences relative to the Input. So my question is what suggestions do you have to see better enrichment in the IP'ed fraction?


Just to be clear, when you run PCR on your Input DNA you dilute it to the same concentration as your IP'd DNA, correct? Otherwise you can't determine enrichment over your input DNA. Or at least you have to calculate using the dilution factor.

In any case, the actual ratio, IP/ Input for a particular genomic location is meaningless unless you compare it to another region where you expect to see very little or no protein X. So regardless of whether your IP/ Input ratio is greater or less than one, it should be higher (for Pol II for instance) at an active gene than at a silent gene. This is what will tell you if your IP is working or not.

-KPDE-