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What's a decent %IP yield?


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#1 SStamis

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Posted 17 September 2010 - 09:38 AM

I have 2 cell lines I am running ChIP on, PC-3 cells and PCa-2b cells. They're human prostate cancer liens. I optimized shearing standards for both of them individually, and got similar IP yields for DNA according to my Nano Drop. But my %IP is incredibly different. When I run QPCR on my gene of interest, PC-3 has a result of 7.2%, while in PCa-2b expression is 0.9%. I am soooooo new to ChIP. Is this okay? I don't want to go on my way thinking I have usable results when in fact I don't. Is optimization necessary? Step to optimize?
Thanks!
Sarah

#2 Dukey

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Posted 17 September 2010 - 04:33 PM

I have 2 cell lines I am running ChIP on, PC-3 cells and PCa-2b cells. They're human prostate cancer liens. I optimized shearing standards for both of them individually, and got similar IP yields for DNA according to my Nano Drop. But my %IP is incredibly different. When I run QPCR on my gene of interest, PC-3 has a result of 7.2%, while in PCa-2b expression is 0.9%. I am soooooo new to ChIP. Is this okay? I don't want to go on my way thinking I have usable results when in fact I don't. Is optimization necessary? Step to optimize?
Thanks!
Sarah


This would depend on several factors. First of all % input values of 0.9% and 7.2% are very good, but this would depend on what you are chipping. If this is a TF, 7.2% is remarkably high and 0.9% is about average. If it is not but is instead histones or something then 0.9% is on the low side probably. I usually get 1-2% for my TF.

I presume you expect the cell lines to be similar but that begs the question, why are you using both of them if they are the same? If they are not the same then you can expect differences. If they are not cultured in the same way, you can expect differences. I personally think the shearing step is where most of the variation lies in chip and you are right to focus on that step.

One thing I would say is that even if the sonication appears similar on a gel, have you really optimised this properly? There is a point in sonication where extra rounds of sonication will not make any difference to the DNA so if your sonication is sufficient at say 4 cycles, but you only check it at 8 cycles, it will look good but in truth it is over-sonicated. You can achieve the same sonication at 4 rounds. This is just an example and in my hands this was a big reason for variation in my chip when I first started out not that long ago.

Finally make sure you are comparing you results to enrichment at a negative control region, ideally another promoter. Arguably this is the most relevant control but is by far the most difficult to achieve. Doing this may shed light on your results. For example, your negative control may be much higher in the PC-3 cells, so the fold change over the negative region is about the same.

Hope this all makes sense, just some rushed thoughts on a Friday night.

#3 SStamis

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Posted 20 September 2010 - 04:45 AM

This would depend on several factors. First of all % input values of 0.9% and 7.2% are very good, but this would depend on what you are chipping. If this is a TF, 7.2% is remarkably high and 0.9% is about average. If it is not but is instead histones or something then 0.9% is on the low side probably. I usually get 1-2% for my TF.

I presume you expect the cell lines to be similar but that begs the question, why are you using both of them if they are the same? If they are not the same then you can expect differences. If they are not cultured in the same way, you can expect differences. I personally think the shearing step is where most of the variation lies in chip and you are right to focus on that step.

One thing I would say is that even if the sonication appears similar on a gel, have you really optimised this properly? There is a point in sonication where extra rounds of sonication will not make any difference to the DNA so if your sonication is sufficient at say 4 cycles, but you only check it at 8 cycles, it will look good but in truth it is over-sonicated. You can achieve the same sonication at 4 rounds. This is just an example and in my hands this was a big reason for variation in my chip when I first started out not that long ago.

Finally make sure you are comparing you results to enrichment at a negative control region, ideally another promoter. Arguably this is the most relevant control but is by far the most difficult to achieve. Doing this may shed light on your results. For example, your negative control may be much higher in the PC-3 cells, so the fold change over the negative region is about the same.

Hope this all makes sense, just some rushed thoughts on a Friday night.



Yes, very helpful. I was worried about over sonication so I'll hone in to that step. Do you think GAPDH is a good control? I'm not sure I know what a good neg control region is. I used an ab for RNA Pol II and that looks good. My gene isn't pulled down by RNA Pol II in either cell line. The reason I have 2 incredibly similar cell lines is that they are from different ethnicities, and this is part of an ethnicity study. The PCa-2b cells are very fickle, and require massive amounts of additives to the culture media. It figures that it would be the difficult cell line to work with in ChIP. anyway, thank you so much Dukey!




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