Dear Bioconductor list, "Design and Analysis of DNA Microarray Investigations" by R. Simons et al. p. 50 states that the Affymetrix spike-in signals should be in the order BioB < BioC < BioD < Cre. I have normalized 38 mouse 430 2.0 arrays with GCRMA 1.10 in Bioconductor 2.0 (I am using the older GCRMA/Bioconductor until I understand the current version better and am sure it is an improvement). The RNA was prepared by 2 rounds of amplification. Most of the arrays follow the above order of spike-in signals: But 3 don't, the worst of which is: AFFX-BioB-3_at 2.294069074 AFFX-BioC-3_at 3.3599997 AFFX-BioDn-3_at 8.462104429 AFFX-CreX-3_at 11.09629022 AFFX-BioB-5_at 3.322136446 AFFX-BioC-5_at 3.093697367 AFFX-BioDn-5_at 8.079313416 AFFX-CreX-5_at 11.06766875 Is the decrease rather than increase between FFX-BioB-5_at and AFFX-BioC-5_at a cause to discard the array. All of the regular affy quality checks, plus the RNA degradation plot and the AffyPLM NUSE ,RLE, and signed residual image for this chip looked fine. THANKS! Rich ------------------------------------------------------------ Richard A. Friedman, PhD Associate Research Scientist, Biomedical Informatics Shared Resource Herbert Irving Comprehensive Cancer Center (HICCC) Lecturer, Department of Biomedical Informatics (DBMI) Educational Coordinator, Center for Computational Biology and Bioinformatics (C2B2)/ National Center for Multiscale Analysis of Genomic Networks (MAGNet) Box 95, Room 130BB or P&S 1-420C Columbia University Medical Center 630 W. 168th St. New York, NY 10032 (212)305-6901 (5-6901) (voice) friedman at cancercenter.columbia.edu http://cancercenter.columbia.edu/~friedman/ "I like it how they tell us that something can either be rational or radical-and this is a college." -Isaac Friedman, age 18