[BioC] HTqPCR useful for ChIP data ?

[Heidi Dvinge]

Hello Guillaume,

I've actualy never thoguht about HTqPCR and ChIP analysis, but that's
solely because I've never received any ChIP-qPCR data myself. I don't see
any a priori reason why you couldn't use the package, at least for QC and
preprocessing.

> Dear Heidi Dvinge
>
> I want to use your package HTqPCR in order to visualize, normalise and
> analyse my rtqPCR data. So before I want to ask you some questions. My
> little dataset is composed of 40 samples. I want to collect data for 8
> genes including 2 housekeeping. The number of genes is very small and I to
> normalize with deltaCt methods. Yet my samples are derived from a ChIP
> experiment. So for all my samples I have 2 extracts, one input, the
> control
> that has not been immunoprecipitated, and an immunoprecipitated
> (IP) extract. PCR is performed on this DNA. The problem is ChIP
> experiment contrain us to have a difference of concentration between Inupt
> and IP, here input is 50 more concentrate than the IP.
>
Just to clarify, what exactly is it you want to test here? Whether there's
a different between Input and IP, or do you have multiple different
treatments, and you want to test whether the IP is more enriched relative
to the Input in some treatment? And with 50x more concentrated, do you
mean the amount of antibody added initially, or the amount of extracted
material?

> So finally I have 2 Ct value for each genes, Input and IP value, and this
> value don't correspond of the same concentration. My question is, do you
> think your tool can be adapted to my case? In the affirmative, do you have
> any suggestion about what to normalize my data between Input and IP Ct
> value ?
>
Normalising can admittedly be a bit tricky when you have so few genes. In
your samples and treatments, can you assume that the two housekeeping
genes ought to have the same signal in IP and Input when adjusted for
concentration? And/or will the level of your six other genes vary across
samples, or are they all from the same treatment?

Best,
\Heidi

> thank you
>
> Guillaume T.
>
> Guillaume Tiberi, ingénieur d'études en Bioinformatique
> guillaume.tiberi at inserm.fr
> 04 91 22 33 33 poste 4186
> Equipe Estelle Duprez
> Centre de Recherche en Cancérologie de Marseille,
> http://crcm.marseille.inserm.fr
> Inserm UMR 891, 27 bd Leï Roure, BP 30059, 13273 Marseille Cedex 09
> France
>