real time PCR with ChIP - (Feb/04/2007 )
Hi
I have been working with ChIP for a few months and the results are quite
random.....sometimes my negetive control (no Ab) works and sometimes a band
appears. I have also tried the quick Chip method and have done both real time PCR and normal
PCR with the DNA.....Although I can get amplification with the input samples
by real time PCR, I have been unable to get amplification of my IP samples.....whereas I
could get amplification for both by normal PCR. Do you quantify the DNA and
use equal amounts of Input, IP and no Ab before you do a PCR? Do you get
amplification in your no Ab control? I am not sure if the problem is with the
ChIP protocol or the real time PCR. I have trieds both the Upstate kit and home made
reagents and have tried blocking beads with BSA us suggested by Upstate.
Any advice will be greatly appreciated.
I have been working with ChIP for a few months and the results are quite
random.....sometimes my negetive control (no Ab) works and sometimes a band
appears. I have also tried the quick Chip method and have done both real time PCR and normal
PCR with the DNA.....Although I can get amplification with the input samples
by real time PCR, I have been unable to get amplification of my IP samples.....whereas I
could get amplification for both by normal PCR. Do you quantify the DNA and
use equal amounts of Input, IP and no Ab before you do a PCR? Do you get
amplification in your no Ab control? I am not sure if the problem is with the
ChIP protocol or the real time PCR. I have trieds both the Upstate kit and home made
reagents and have tried blocking beads with BSA us suggested by Upstate.
Any advice will be greatly appreciated.
I'm assuming that you are getting fairly low Ct for your input samples. If you are getting high Ct with Input samples, could it be possible that your primers are not ideal for doing real time? Another possibility: Is it possible that there is a contaminant in your IP DNA which is incompatible with the master mix you are using for real time?
As for your question of seeing a signal in your no Ab control, we regularly get signal in our mock IP whether we use pre-immune IgG or no antibody at all. We don't worry about this since we quantitate our results as the ratio of specific IP signal to mock IP signal and compare the region of interest to a region where the factor of interest doesn't bind (or at least only binds at low level).
It sounds like you've already tried a few protocols but I just thought I'd suggest ours. It takes about 4 hours or less when starting with sonicated chromatin to do IP and get PCR ready DNA. It's here at Nature Protocols: http://www.nature.com/nprot/journal/v1/n1/...ot.2006.27.html
I'm assuming that you are getting fairly low Ct for your input samples. If you are getting high Ct with Input samples, could it be possible that your primers are not ideal for doing real time? Another possibility: Is it possible that there is a contaminant in your IP DNA which is incompatible with the master mix you are using for real time?
As for your question of seeing a signal in your no Ab control, we regularly get signal in our mock IP whether we use pre-immune IgG or no antibody at all. We don't worry about this since we quantitate our results as the ratio of specific IP signal to mock IP signal and compare the region of interest to a region where the factor of interest doesn't bind (or at least only binds at low level).
It sounds like you've already tried a few protocols but I just thought I'd suggest ours. It takes about 4 hours or less when starting with sonicated chromatin to do IP and get PCR ready DNA. It's here at Nature Protocols: http://www.nature.com/nprot/journal/v1/n1/...ot.2006.27.html
[/quote]
Thanks for your suggestions.
The Ct values for the input samples are fine but there is no amplification for th IP samples. Is 2 ul of ChIP ed DNA suficient for QPCR?
Its is a relief to know that most people have the background problem. However how do you represent your input sample data? Do you take this into consideration when calculating your ratio between mock IP and specific IP?
I have tried the protocol that you suggested and certainly it saves a lot of time. However although I got higher concentrations of DNA, the DNA was not very pure. Do you further perform a PCI extraction? and what quantity do you use for the real time PCR?
Thanks again
As for your question of seeing a signal in your no Ab control, we regularly get signal in our mock IP whether we use pre-immune IgG or no antibody at all. We don't worry about this since we quantitate our results as the ratio of specific IP signal to mock IP signal and compare the region of interest to a region where the factor of interest doesn't bind (or at least only binds at low level).
It sounds like you've already tried a few protocols but I just thought I'd suggest ours. It takes about 4 hours or less when starting with sonicated chromatin to do IP and get PCR ready DNA. It's here at Nature Protocols: http://www.nature.com/nprot/journal/v1/n1/...ot.2006.27.html
Thanks for your suggestions.
The Ct values for the input samples are fine but there is no amplification for th IP samples. Is 2 ul of ChIP ed DNA suficient for QPCR?
Its is a relief to know that most people have the background problem. However how do you represent your input sample data? Do you take this into consideration when calculating your ratio between mock IP and specific IP?
I have tried the protocol that you suggested and certainly it saves a lot of time. However although I got higher concentrations of DNA, the DNA was not very pure. Do you further perform a PCI extraction? and what quantity do you use for the real time PCR?
Thanks again
1) Regarding background, some people will do a number of things to try to bring the backround down successfully. Blocking the beads with salmon sperm DNA and/ or BSA, reducing the amount of beads used, and diluting the chromatin further before IPing are all things others have used. In addition, more stringent washes may help (although this has had little effect in my hands).
2) As to your question of how we represent our data, we express it as a ratio of specific to background signal as follows:
2^(Ct of mock IP - Ct of specific IP)
3) You are correct that, using our fast method, the DNA is not very pure. This is not a problem for PCR analysis but is certainly an issue if you are doing ChIP on chip. In this case others have done mini-elute clean-ups before doing the amplification.
4) When you load IPed DNA from our fast protocol we recomend you load enough template to make up 25 to 50% of the total reaction volume. We use the ABI 7900 thermocycler and SensiMix master mix from Quantace, although we've used ABI's master mix as well and it's worked fine.
Hope that helps,
Joel
I will try what you suggested. Thanks a lot!