ChIP: Equal DNA Pull-Down in Positive and Negative Controls - HELP - (Sep/11/2006 )
I have been working with ChIP for about a month now. I have done 5 separate experiments and have run into the same complication each time. My negative control antibody (anti-GFP) seems to be pulling down the same amount of DNA as my positive control (anti-RNA Pol II). Each IP should have roughly 2 million cells and I’m following the upstate EZ-ChIP protocol. I’m analyzing my pull-down using qPCR with the GAPDH primers supplied by Upstate.
The sonications look good and I’m purifying my DNA by phenol:chloroform extraction. After an overnight stay in the -20 and a 15min spin at 14,000rpm there is a very small streak visible in my input tube and no visible streak in my IP tubes.
Upon qPCR analysis the Ct value for my input (10% volume of the IP) is 28, the Ct value for RNAP is 32, and the Ct value for GFP is 33.
The gene is lowly expressed in the cell line I’m working in(which might explain a low level of pull-down using the RNAP antibody) however I feel that the difference in Ct value for my positive and negative control should be more than 1. I’ve heard that this difference should be between 8-10 cycles (but that’s just hearsay).
Has anyone encountered a problem like this and if so are there any easy solutions?
The sonications look good and I’m purifying my DNA by phenol:chloroform extraction. After an overnight stay in the -20 and a 15min spin at 14,000rpm there is a very small streak visible in my input tube and no visible streak in my IP tubes.
Upon qPCR analysis the Ct value for my input (10% volume of the IP) is 28, the Ct value for RNAP is 32, and the Ct value for GFP is 33.
The gene is lowly expressed in the cell line I’m working in(which might explain a low level of pull-down using the RNAP antibody) however I feel that the difference in Ct value for my positive and negative control should be more than 1. I’ve heard that this difference should be between 8-10 cycles (but that’s just hearsay).
Has anyone encountered a problem like this and if so are there any easy solutions?
I actually have encountered a problem like that but the problem in my case was that the input chromatin was too concentrated. With the Cts you're getting that doesn't seem to be the problem. With the 4 CT difference between input and IP I would say you're getting good pulldown as well (about 0.6%) so I don't think the antibody is dead.
Some suggestions I would have would be to use a different control primer than the one you're using (like for b-actin or some other highly expressed housekeeping gene) on the ChIPs you've already done.
If you plan to try another ChIP I would use a different control antibody like pre-immune IgG (we use Vector Labs' cat# I-1000) or just beads alone. There's no chance that your cells are expressing a GFP fusion protein, is there?
Joel
There is no way GFP is being expressed in my cells.
However, I just checked my input DNA and realized that I have been doing my qPCR with 600ng of DNA. My pull-downs also have much more DNA than I had originally thought.
I tried qPCR with a 2 fold dilution of my sample (before I realized how concentrated it was) and loading only 300ug of DNA gave me a Ct of 22 (a 6 cycle decrease much to my surprise).
I think all of my samples have too much DNA so I'm doing serial dilutions of my samples to see if I can get better Ct resolution.
Thanks for the input and I'll let you know how it goes.
However, I just checked my input DNA and realized that I have been doing my qPCR with 600ng of DNA. My pull-downs also have much more DNA than I had originally thought.
I tried qPCR with a 2 fold dilution of my sample (before I realized how concentrated it was) and loading only 300ug of DNA gave me a Ct of 22 (a 6 cycle decrease much to my surprise).
I think all of my samples have too much DNA so I'm doing serial dilutions of my samples to see if I can get better Ct resolution.
Thanks for the input and I'll let you know how it goes.
If diluting your input and IP'd DNA doesn't change anything then I would try dilutions of your chromatin in parallel ChIPs (maybe 4x to start with). This may decrease your background enough to see the signal over the background.
Maybe try doing more washes.
We do 8-10 washes to get rid of background
when doing qPCR analyses.
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 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. Do you have any idea how I can solve this potential problem?
Styounger did you ever get better results from this assay?
I have gotten better results, however I have stopped using the EZ ChIP kit. I used a protocol that can be found at http://www.epigenome-noe.net/researchtools...l.php?protid=28. Its important to get good quality reagents, especially Protein A/G beads. I believe the low quality of the EZ ChIP kit components plays a role in the poor results I was getting. With the kit my Ct values for negative control IgG and RNA Pol II were the same. With the nicer reagents I get nearly a 10 Ct difference. Let me know if you have any questions.
Styounger,
I continue to get high backgrounds and I've tried all the tricks and techniques from more/longer washing to pre-blocking. I am using magnetic beads. Any opinions on the magnetic vs. agarose bead for ChIPs? Thx.
I continue to get high backgrounds and I've tried all the tricks and techniques from more/longer washing to pre-blocking. I am using magnetic beads. Any opinions on the magnetic vs. agarose bead for ChIPs? Thx.
I've never used the magnetic beads, so I can't really comment on that. One option, which I'm not a huge fan of, is clearing your sample by incubation with negative control antibody for an hour or so. Then use beads to clear out the antibody and also minimize non-specific binding to the beads. Take your cleared sample and resume your regular ChIP protocol. Have you tried this?