Dear all, Thanks for your answer about my design problem. I have another question about the RNA degradation control of Affy array. With the Affymetrix software (in the expression report), the controls indicated there was no RNA degradation in my arrays. But when I analyse the AffyBatch with R, I have highly significant difference between 5' and 3' also observed in the RNA digestion plot, so degradation. Can you explain the difference between the two observations with Affy software and affy package of Bioconductor ? And which control can I trust ? Thanks in advance. Delphine -- ================================================================== Delphine Fleury DEPARTMENT OF PLANT SYSTEMS BIOLOGY Fax:32 (0)9 3313809 GHENT UNIVERSITY/VIB, Technology Park 927, B-9052 Gent, Belgium Vlaams Interuniversitair Instituut voor Biotechnologie VIB mailto:defle@psb.ugent.be http://www.psb.ugent.be

The controls that Affy uses are spiked-in transcripts that are designed to indicate problems in the hybridization step, which would be indicative of a 'bad' chip. The RNA digestion plot gives a qualitative idea of two things; the amount of RNA degradation that occurred during the preparation of your RNA (due presumably to RNAases), and how well the second strand synthesis went in the preparation of your samples. Since these are two completely different things, you cannot compare the results and ask which one you should believe. You can believe both and there is no contradiction. As an aside, I almost always see a 'significant' slope to the curves in the RNA digestion plot. I don't necessarily think this is a critical problem. However, if the curves have completely different slopes, I start to get worried (not that there is much I can do at that late stage...). Jim James W. MacDonald Affymetrix and cDNA Microarray Core University of Michigan Cancer Center 1500 E. Medical Center Drive 7410 CCGC Ann Arbor MI 48109 734-647-5623 >>> defle@psb.ugent.be 08/22/03 11:07AM >>> Dear all, Thanks for your answer about my design problem. I have another question about the RNA degradation control of Affy array. With the Affymetrix software (in the expression report), the controls indicated there was no RNA degradation in my arrays. But when I analyse the AffyBatch with R, I have highly significant difference between 5' and 3' also observed in the RNA digestion plot, so degradation. Can you explain the difference between the two observations with Affy software and affy package of Bioconductor ? And which control can I trust ? Thanks in advance. Delphine -- ================================================================== Delphine Fleury DEPARTMENT OF PLANT SYSTEMS BIOLOGY Fax:32 (0)9 3313809 GHENT UNIVERSITY/VIB, Technology Park 927, B-9052 Gent, Belgium Vlaams Interuniversitair Instituut voor Biotechnologie VIB mailto:defle@psb.ugent.be http://www.psb.ugent.be _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor