[BioC] using genomic DNA as universal reference

[oliveros at cnb.csic.es]

Dear Jin,

I used to work with this kind of data in the past: RNA in one channel and
genomic DNA (gDNA) in the other. We used the gDNA as a reference value for
each gene to quantify the amount of DNA present in each spot.

I also noticed that the distribution of the intensities was different in
both types of samples. In fact this is expectable as the amount of mRNA
molecules in one sample has nothing to do with the amount of gDNA for the
same gene in other sample.

So I normalized the data separately:

-I created two tables, one with all RNA values and other with all gDNA
values.

-I adjusted the quantiles of each table separately.

-Then I calculated the ratio RNA intensity / gDNA intensity and I used
this ratio RNA/gDNA as the expression value of the genes. In further
analysis steps I treated them as data coming from single channel
hybridizations.


I hope that helps.

best,

Juan Carlos Oliveros
Head of BioinfoGP Unit at CNB-CSIC
Madrid, Spain
http://bioinfogp.cnb.csic.es


> Dear list,
>
> I would like to ask comments and suggestions on how to normalize
> microarray
> data with genomic DNA as reference.
>
> The experiments were performed with bacterial RNA and genomic DNA samples.
> What I noticed was that the data were pretty consistent across all chips
> on
> both channels.  But there exists a huge difference between the two
> channels
> in terms of the distribution of the probe intensities, although the
> average
> intensities were the same for the both channels. T statistics with
> non-normalized data showed that there were two thirds probes with p values
> <= 0.05 by comparing the hybridization intensities between red and green
> channels.
>
> Regarding to the huge difference described above the normalization methods
> people usually use may not be appropriate for the RNA/DNA data sets. What
> normalization algorithms would be useful if there is any? Does anyone have
> experience with this?
>
> Any comments or suggestions will be appreciated!
>
> Jianping Jin
>
>
> ##################################
> Jianping Jin Ph.D.
> Bioinformatics scientist
> Center for Bioinformatics
> Room 3133 Bioinformatics building
> CB# 7104
> University of Chapel Hill
> Chapel Hill, NC 27599
> Phone: (919)843-6105
> FAX:   (919)843-3103
> E-Mail: jjin at email.unc.edu
>
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