[BioC] Question about normalization of microarray data

[Johan Lindberg]

Hi all. I have a question about normalization of microarray data.
 
In our lab we use in-house spotted cDNA arrays. We have so far used
commercial reference when doing reference design. We are now trying a
new approach but we have problems with normalization. What we have done
is to pool product from every spot on the chip and done in vitro
transcription on the PCR product. So we have RNA corresponding to every
spot on the chip. Then this is used as a reference. It is much cheaper
and we get signal from every spot on the chip instead of having spots
with no signal in both channels. But when one looks at an MA-plot the
plot will be skewed towards the reference. There are about (in this
pilot case) 2000 spots that only give signal in the reference channel
(which will skew the MA-plot). This will make many assumptions not
correct when normalizing the data, e.g. using lowess normalization
assuming that the ratio R/G should be 1 for most spots. 
Since the case for this kind of data is that one channel should be much
stronger than the other, and we want to keep the normalization within
slide (to be able to correct for spatial biases and intensity dependent)
the only way I could think of is by spotting a lot of control spots (not
present in the tested RNA or the reference RNA) and use these to
normalize the data. 
 
Any comments of tips of how to normalize this kind of data are greatly
appreciated.
 
Best regards 
 
//Johan Lindberg
 
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Johan Lindberg
Royal Institute of Technology
AlbaNova University Center
Stockholm Center for Physics, Astronomy and Biotechnology
Department of Molecular Biotechnology
106 91 Stockholm, Sweden
 
Phone (office) +46 8 553 783 44
Fax + 46 8 553 784 81
Visiting adress Roslagstullsbacken 21, Floor 3
Delivery adress Roslagsvägen 30B
http://www.biotech.kth.se/molbio/microarray/index.html
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