[BioC] Affy vs. cDNA : Low and not expressed genes

[Adaikalavan Ramasamy]

Dear all,

Thank you for the very interesting discussion on the topic of
"replicates and low expression levels" in the last few day. I am facing
a related problem regarding normalization and would appreciate any
advice.

A small time course experiment was done on blood macrophages and
hybridized to affymetrix chip HGU-133A. There were 3 replicates and 3
time points (0, 2, 48 hour).

The main problem is that at time point 0 hour, there are 95 % Absent
calls. The percentage of Absent call decreases to 70% in 2 hour and 20%
in 2 days. Initially I assumed that there was some physical problem with
the array. But later I was corrected by the biologists that it was
expected as many genes are not expressed in blood macrophages. Thus most
of the 95 % Absent were due to not expressed genes ... Apparently this
is common in developmental biology.

My first question is how does one normalize this kind of data ? The
assumption in two-colour cDNA data of "most of the genes are not
differently expressed" does not hold here. Median normalization would
not be meaningful in this scenario.

We then explored the possibility of using housekeeping genes for
normalization. But it seems that the 100 housekeeping genes for HGU-133A
are standard and not specified for our experiment. This is because only
28 of these 100 genes are expressed through out all time points.


The biologists have decided to re-do the experiment again and I think
they are more likely to hear our advice BEFORE doing the experiments. My
second question is this: Will 2-colour cDNA with UHR as reference
overcome this problem ?

Now I would expect to see most of the un-expressed genes (and previously
Absent in affy) to have very negative log ratio values. But I don't
think the assumption of "most genes are not differentially expressed"
will hold again. And how does one deal with this ... 

My last question is has anyone done a comparison of Affymetrix to cDNA
results/efficiency/advantages. I am interested in quantifying the
benefits of spending 5 times as much money on something that has
typically 40% absent calls. Thank you very much in advance.

Regards, Adai.