[BioC] RNA degradation and no diff expressed genes

[brini.elena at hsr.it]

Dear all,
I have a problem with the chips I did recently.
I checked the quality of RNA extracted using Trizol from human PBMC 
and it was not so bad (RIN 7-9).
I checked the profile of cRNA after IVT using One-cycle kit of 
Affymetrix and it peaked around 1000 nucleotides.
Then I hybridized the human U133 Plus 2 chips and the % of Present was 
around 38-40%. I did the Affy QC and it showed a problem of RNA 
degradation in almost all the chips (26): the 3’/5’ ratio is not 
inside the correct range. Also in the Poly-A 5’ Probes plot I have 
some problems.
Moreover the average signal intensity of P in each chip, using a TGT 
value of 100, is around 400. Is it normal or is it very low?
When we performed the analysis (Limma), I could not find 
differentially expressed genes, I mean I found around 50 genes and I 
know this is not the case, because I did similar experiments in the 
past and I always found around 1000 of genes!
How can be all this problems linked together?
How can I have an expected % of Present of 40 and no genes 
differentially expressed?
I think I had problems during amplification/labeling but I still 
cannot understand in which passage.

Any help?

Thanks a lot.
Elena