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[Sample prep] Enrichment and aspecific control use with qPCR

[Sample prep] Enrichment and aspecific control use with qPCR - ChIP Chromatin Immunoprecipitation Forum

[Sample prep] Enrichment and aspecific control use with qPCR - Chromatin Immunoprecipitation Forum. Discuss, chat about, and post questions about the ChIP assay, in the ChIP forum. Includes troubleshooting, protocols, buffer preparation, and more.


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Old 02-12-2009, 08:04 AM
Pipette Filler
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Default [Sample prep] Enrichment and aspecific control use with qPCR



Hello,

I am interest in doing a ChIP for ChIP-seq. As a control I have seen that DNA concentrations with qPCR and specific primers are determined. From these concentrations is an enrichment value calculated. Before the enrichment value is calculated, many times people correct the concentration of the samples for the input concentration. But what I also see is that some people are using a control for aspecific binding (Rabbit IgG control). But is this a background sample, because with background I think of an addition to my sample value like in spectrometry (and in your sample is no IgG antibody present only the target antibody).
So results this in concentration target antibody sample - concentration IgG sample?
or in concentration target antibody sample/ concentration IgG sample?
And how good is IgG as a control? Is it really necessary to use an a specific binder? And what does it say about mine ChIP? Can you use it as a ChIP to ChIP variation value (the difference between the enrichment's).

A lot of questions, I am really curious to your opinions.
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Old 04-29-2009, 05:38 PM
Pipette Filler
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Default Re: [Sample prep] Enrichment and aspecific control use with qPCR

Hi Danny, snap, I'm trying to get a ChIP-Seq protocol going as well.

In response to your many questions, the answer does rather depend on the question you are asking. But for very good reasons explained in the paper cited below I have always used %input to normalise ChIP data unless the experimental design prevents me from doing. This is slowly becoming convention in labs that use ChIP a lot but yes, I agree that there is a lot of variation in the literature as to what normalisation method is used.

Haring M, Offermann S, Danker T, Horst I, Peterhaensel P & Stam M. 2007. Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization. Plant Methods, 3:11.
(don't be freaked out it's a plant ChIP paper, the arguments apply to any ChIP).
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Old 05-08-2009, 09:44 AM
Pipette Filler
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Default Re: [Sample prep] Enrichment and aspecific control use with qPCR

The problem with using unspecific IgG as "negative control" is that the binding behaviour of these IgG may differ more or less from this of your specific antibody
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Old 05-09-2009, 08:22 AM
Pipette Filler
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Default Re: [Sample prep] Enrichment and aspecific control use with qPCR

Quote:
Originally Posted by Joegreen View Post
The problem with using unspecific IgG as "negative control" is that the binding behaviour of these IgG may differ more or less from this of your specific antibody
I have the same thoughts about this. Some suggest a sample without antibody as negative control and/or a sample with DNA of which is known the antibodiestarget is not available, however this is not always possible
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aspecific , control , enrichment , igg control , prep , qpcr , sample


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