10 replies to this topic

[jangajarn]

Hi all,
I'm trying to do ChIP and use the resulting DNA to do a qPCR. But the problem is that the amount of DNA in ChIP is very low that I do not get any signal once I do the qPCR. So anybody has suggestions to improve the amount of DNA? I tried doing a PCR of the ChIP product with random hexamers to amplify the signal, but its also in vain. Any suggestions welcome.
Thanks so much!

[macroman]

I believe that rather than struggling with improving at the end point. Its always a good idea to improve the starting parameters. More over we perform qPCR of the Chip products for quantitative data only after observing the initial PCR products on gel.

Hope this helps....

[KPDE]

jangajarn, on Sep 28 2009, 01:44 PM, said:

Hi all,
I'm trying to do ChIP and use the resulting DNA to do a qPCR. But the problem is that the amount of DNA in ChIP is very low that I do not get any signal once I do the qPCR. So anybody has suggestions to improve the amount of DNA? I tried doing a PCR of the ChIP product with random hexamers to amplify the signal, but its also in vain. Any suggestions welcome.
Thanks so much!

As long as your primers are working and have a good efficiency (use your input samples to determine this) then what you need to optimize is the ChIP assay.  You shouldn't have to amplify your samples before doing PCR just to get a signal.

Are you doing ChIP with a positive control antibody.  This is an antibody that many have used successfully in ChIP and which gives unambiguous results.   The 4H8 monoclonal for the Pol II CTD (Santa Cruz sc-47701 or Abcam ab5408) works really well in ChIP and can give you clear results if you have an inducible gene (i.e. you should see higher Pol II density after inducing the gene).  Even at steady state, most expressed genes have a higher level of Pol II just downstream of the TSS than in the rest of the gene or upstream of the TSS so you'll have a pattern to look for to tell if your ChIP is working.  Optimize ChIP using a positive control antibody like this.

[jangajarn]

macroman, on Sep 28 2009, 02:06 PM, said:

I believe that rather than struggling with improving at the end point. Its always a good idea to improve the starting parameters. More over we perform qPCR of the Chip products for quantitative data only after observing the initial PCR products on gel.

Hope this helps....

Thats exactly my problem. We are not able to visualize the ChIP products. Thats why we tried doing PCR of the CHiP product hoping that we would increase the yield of the product DNA (with random hexamers). Did you have to do PCR at all for visualization?

[jangajarn]

KPDE, on Sep 28 2009, 05:01 PM, said:

As long as your primers are working and have a good efficiency (use your input samples to determine this) then what you need to optimize is the ChIP assay.  You shouldn't have to amplify your samples before doing PCR just to get a signal.

Are you doing ChIP with a positive control antibody.  This is an antibody that many have used successfully in ChIP and which gives unambiguous results.   The 4H8 monoclonal for the Pol II CTD (Santa Cruz sc-47701 or Abcam ab5408) works really well in ChIP and can give you clear results if you have an inducible gene (i.e. you should see higher Pol II density after inducing the gene).  Even at steady state, most expressed genes have a higher level of Pol II just downstream of the TSS than in the rest of the gene or upstream of the TSS so you'll have a pattern to look for to tell if your ChIP is working.  Optimize ChIP using a positive control antibody like this.

Thanks for the reply.. we can see the products through qPCR (testde through a positive control to RNA polII). But our problem is that we want to visualize the CHiP products on a gel before going to QPCR. Do you have any suggestions?

[KPDE]

jangajarn, on Sep 30 2009, 01:17 PM, said:

KPDE, on Sep 28 2009, 05:01 PM, said:

As long as your primers are working and have a good efficiency (use your input samples to determine this) then what you need to optimize is the ChIP assay.  You shouldn't have to amplify your samples before doing PCR just to get a signal.

Are you doing ChIP with a positive control antibody.  This is an antibody that many have used successfully in ChIP and which gives unambiguous results.   The 4H8 monoclonal for the Pol II CTD (Santa Cruz sc-47701 or Abcam ab5408) works really well in ChIP and can give you clear results if you have an inducible gene (i.e. you should see higher Pol II density after inducing the gene).  Even at steady state, most expressed genes have a higher level of Pol II just downstream of the TSS than in the rest of the gene or upstream of the TSS so you'll have a pattern to look for to tell if your ChIP is working.  Optimize ChIP using a positive control antibody like this.

Thanks for the reply.. we can see the products through qPCR (testde through a positive control to RNA polII). But our problem is that we want to visualize the CHiP products on a gel before going to QPCR. Do you have any suggestions?

What kind of Ct are you getting with qPCR?  In my experience, if I got amplification in qPCR then I could always see the products on a gel.

[chrismbyrd]

jangajarn, on Sep 30 2009, 04:17 PM, said:

KPDE, on Sep 28 2009, 05:01 PM, said:

As long as your primers are working and have a good efficiency (use your input samples to determine this) then what you need to optimize is the ChIP assay.  You shouldn't have to amplify your samples before doing PCR just to get a signal.

Are you doing ChIP with a positive control antibody.  This is an antibody that many have used successfully in ChIP and which gives unambiguous results.   The 4H8 monoclonal for the Pol II CTD (Santa Cruz sc-47701 or Abcam ab5408) works really well in ChIP and can give you clear results if you have an inducible gene (i.e. you should see higher Pol II density after inducing the gene).  Even at steady state, most expressed genes have a higher level of Pol II just downstream of the TSS than in the rest of the gene or upstream of the TSS so you'll have a pattern to look for to tell if your ChIP is working.  Optimize ChIP using a positive control antibody like this.

Thanks for the reply.. we can see the products through qPCR (testde through a positive control to RNA polII). But our problem is that we want to visualize the CHiP products on a gel before going to QPCR. Do you have any suggestions?

Hello KPDE,

Are you trying to visualize the IP itself or the PCR of the IP?  I have never done it, but would bet that (if you could actually see it) it would be a small smear of DNA similar to the smear of whole genomic DNA you start with.  What would that tell you?

Edited by chrismbyrd, 19 October 2009 - 10:40 AM.

[giny]

KPDE, on Sep 30 2009, 03:35 PM, said:

jangajarn, on Sep 30 2009, 01:17 PM, said:

KPDE, on Sep 28 2009, 05:01 PM, said:

As long as your primers are working and have a good efficiency (use your input samples to determine this) then what you need to optimize is the ChIP assay.  You shouldn't have to amplify your samples before doing PCR just to get a signal.

Are you doing ChIP with a positive control antibody.  This is an antibody that many have used successfully in ChIP and which gives unambiguous results.   The 4H8 monoclonal for the Pol II CTD (Santa Cruz sc-47701 or Abcam ab5408) works really well in ChIP and can give you clear results if you have an inducible gene (i.e. you should see higher Pol II density after inducing the gene).  Even at steady state, most expressed genes have a higher level of Pol II just downstream of the TSS than in the rest of the gene or upstream of the TSS so you'll have a pattern to look for to tell if your ChIP is working.  Optimize ChIP using a positive control antibody like this.

Thanks for the reply.. we can see the products through qPCR (testde through a positive control to RNA polII). But our problem is that we want to visualize the CHiP products on a gel before going to QPCR. Do you have any suggestions?

What kind of Ct are you getting with qPCR?  In my experience, if I got amplification in qPCR then I could always see the products on a gel.

Sigh....I have been struggling with ChIP assay for months!! After ChIP, i supposed to run qPCR to check the products. But the CT i got was in the range of 26 to 32, very low....if i increase the DNA template for qPCR, all I can see is noise, no CT at all. Can anyone tell me what i should do with that?

[Radish]

giny, on Nov 9 2009, 03:11 AM, said:

KPDE, on Sep 30 2009, 03:35 PM, said:

jangajarn, on Sep 30 2009, 01:17 PM, said:

KPDE, on Sep 28 2009, 05:01 PM, said:

As long as your primers are working and have a good efficiency (use your input samples to determine this) then what you need to optimize is the ChIP assay.  You shouldn't have to amplify your samples before doing PCR just to get a signal.

Are you doing ChIP with a positive control antibody.  This is an antibody that many have used successfully in ChIP and which gives unambiguous results.   The 4H8 monoclonal for the Pol II CTD (Santa Cruz sc-47701 or Abcam ab5408) works really well in ChIP and can give you clear results if you have an inducible gene (i.e. you should see higher Pol II density after inducing the gene).  Even at steady state, most expressed genes have a higher level of Pol II just downstream of the TSS than in the rest of the gene or upstream of the TSS so you'll have a pattern to look for to tell if your ChIP is working.  Optimize ChIP using a positive control antibody like this.

Thanks for the reply.. we can see the products through qPCR (testde through a positive control to RNA polII). But our problem is that we want to visualize the CHiP products on a gel before going to QPCR. Do you have any suggestions?

What kind of Ct are you getting with qPCR?  In my experience, if I got amplification in qPCR then I could always see the products on a gel.

Sigh....I have been struggling with ChIP assay for months!! After ChIP, i supposed to run qPCR to check the products. But the CT i got was in the range of 26 to 32, very low....if i increase the DNA template for qPCR, all I can see is noise, no CT at all. Can anyone tell me what i should do with that?

Hello everyone

I have been trying ChIP for four months now with no success at all.
I also had the same problem, my ct values were to the roof, so I decided to change to the fast chip protocol (I figured that a simpler protocol would ultimately reduce the amount of mistakes that I could make), and now I can't even get DNA   .
Maybe I chose the wrong career, or I am completely stupid.

[KPDE]

Radish, on Nov 24 2009, 05:45 PM, said:

giny, on Nov 9 2009, 03:11 AM, said:

KPDE, on Sep 30 2009, 03:35 PM, said:

jangajarn, on Sep 30 2009, 01:17 PM, said:

KPDE, on Sep 28 2009, 05:01 PM, said:

As long as your primers are working and have a good efficiency (use your input samples to determine this) then what you need to optimize is the ChIP assay.  You shouldn't have to amplify your samples before doing PCR just to get a signal.

Are you doing ChIP with a positive control antibody.  This is an antibody that many have used successfully in ChIP and which gives unambiguous results.   The 4H8 monoclonal for the Pol II CTD (Santa Cruz sc-47701 or Abcam ab5408) works really well in ChIP and can give you clear results if you have an inducible gene (i.e. you should see higher Pol II density after inducing the gene).  Even at steady state, most expressed genes have a higher level of Pol II just downstream of the TSS than in the rest of the gene or upstream of the TSS so you'll have a pattern to look for to tell if your ChIP is working.  Optimize ChIP using a positive control antibody like this.

Thanks for the reply.. we can see the products through qPCR (testde through a positive control to RNA polII). But our problem is that we want to visualize the CHiP products on a gel before going to QPCR. Do you have any suggestions?

What kind of Ct are you getting with qPCR?  In my experience, if I got amplification in qPCR then I could always see the products on a gel.

Sigh....I have been struggling with ChIP assay for months!! After ChIP, i supposed to run qPCR to check the products. But the CT i got was in the range of 26 to 32, very low....if i increase the DNA template for qPCR, all I can see is noise, no CT at all. Can anyone tell me what i should do with that?

Hello everyone

I have been trying ChIP for four months now with no success at all.
I also had the same problem, my ct values were to the roof, so I decided to change to the fast chip protocol (I figured that a simpler protocol would ultimately reduce the amount of mistakes that I could make), and now I can't even get DNA   .
Maybe I chose the wrong career, or I am completely stupid.

Don't worry about it.  You're not in the wrong career just because you can't get ChIP to work.  I've had the method stop working for unexplained reasons even after I'd been doing it successfully for years.  Are you still having troubles getting the input DNA?

[Radish]

Thank you very much. I am going for another round today, maybe today is the day.
I just feel frustrated