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