First ChIP - good start? - (Feb/10/2010 )
OK briefly guys.
Introduction
Just started working on ChIP. I am using a combination of Magnify Chip (Invitrogen) and Nelson et al.(2006) protocols. I am using the rat hepatoma cell line, H-4-II-E. My initial IP was done with anti-Pol II (sc899) on about 200,000 cells using 1ug of antibody during an only 2hr incubation in the cold room. I then performed qRT-PCR using primers to rat GAPDH promotor region as a positive control and primers to the GLUT4 coding sequence as my negative control region since it should be silent in liver. Sonication looks nice with a good smear from around 700 - 250bp.
Results (Ct) (Figure 1):
GAPDH
Input (10%) - 23.3
Pol II Ab - 26.8
Mock IgG - undetectable
GLUT4
Input - 23.0
Pol II Ab - 28.4
Mock IgG - undetectable
Interpretation:
By my calculations, this only gives me an approx 1% of input for GAPDH but this is approximately 3.5 fold higher than the % input for the negative control GLUT4 region. I expected the GLUT4 to be almost undetectable but I am encouraged that my mock is non-existent, although I think this is not very meaningful.
My questions are:
- 1% input for Pol II at GAPDH seems low but is this OK for basal conditions?
- Do I have something to go on here? i.e. is it worth me now optimizing time, concentration of antibody etc to fine tune this?
- Could the fact that I am working on rat tissue reduce the efficiency of this particular Pol II Ab? Anbody have any experience with this.
- Is my negative control GLUT4 region OK? Should I perhaps re-design the primers for an intronic region? I did not think it really mattered since there really should be no mRNA expressed in the liver for GLUT4. However, I am not familiar with Pol II binding behaviour.
Any comments welcome.
I'd say it's a good start. I've run into similar issues with using a negative control region (as you know from responding to one of my previous posts...thanks btw!). One of the issues, I think, with using PolII and looking at a supposed negative control region, is that there is always the possibility of ncRNA or some other crazy stuff that is getting transcribed, even if the gene itself isn't expressed. I've struggled with trying to figure out good control regions, but am at a bit of a loss. For other TFs I've been thinking of using a primer towards LINE elements, but that likely wouldn't work for PolII.
I agree with you that it is difficult to interpret the mock data, although it appears to be a popular method to use for a comparison, or "proof" of TF binding in the literature. If only a perfect control for ChIP existed!
MM
Dukey on Feb 10 2010, 02:35 PM said:
Introduction
Just started working on ChIP. I am using a combination of Magnify Chip (Invitrogen) and Nelson et al.(2006) protocols. I am using the rat hepatoma cell line, H-4-II-E. My initial IP was done with anti-Pol II (sc899) on about 200,000 cells using 1ug of antibody during an only 2hr incubation in the cold room. I then performed qRT-PCR using primers to rat GAPDH promotor region as a positive control and primers to the GLUT4 coding sequence as my negative control region since it should be silent in liver. Sonication looks nice with a good smear from around 700 - 250bp.
Results (Ct) (Figure 1):
GAPDH
Input (10%) - 23.3
Pol II Ab - 26.8
Mock IgG - undetectable
GLUT4
Input - 23.0
Pol II Ab - 28.4
Mock IgG - undetectable
Interpretation:
By my calculations, this only gives me an approx 1% of input for GAPDH but this is approximately 3.5 fold higher than the % input for the negative control GLUT4 region. I expected the GLUT4 to be almost undetectable but I am encouraged that my mock is non-existent, although I think this is not very meaningful.
My questions are:
- 1% input for Pol II at GAPDH seems low but is this OK for basal conditions?
- Do I have something to go on here? i.e. is it worth me now optimizing time, concentration of antibody etc to fine tune this?
- Could the fact that I am working on rat tissue reduce the efficiency of this particular Pol II Ab? Anbody have any experience with this.
- Is my negative control GLUT4 region OK? Should I perhaps re-design the primers for an intronic region? I did not think it really mattered since there really should be no mRNA expressed in the liver for GLUT4. However, I am not familiar with Pol II binding behaviour.
Any comments welcome.
I think you might be able to decrease your chromatin at least 2-fold in order to get a better %input and possibly a larger difference from your negative control region. Also, I've found intergenic regions 10 or more kb away from any gene make really good negative controls. Out of curiosity, what is the hepatoma cell line you're using?
Joel
Mighty Mouse is correct in terms of pol2 enriching to regions where the mechanisms are still unclear, like ncRNA. you may also consider titrating the antibody although your data looks great. alternatively, 2hrs incubation could be cut back to 1hr. :-)
Thanks guys, great comments! I will try reducing the chromatin amount and will probably titrate the antibody a little bit.
KPDE - I am using the H-4-II-E cell line (rat). It is a great line that has some key features for us, like its ability to secrete glucose (gluconeogenesis) and it's response to physiological concentrations of insulin. The only downside is it is difficult to transfect with 5% transfection rate about the norm with normal methods. We use electroporation though (Neon) and can get close to 100%.