Dear BioConductor mailing list! I perform 2 color hybridization with Exiqon slides, where each mir has 4 replicates. I compare a rather small number of slides (from 2 up to 6 biologial replicates for each treatment). For analysis of the data I am using the R-2.8, the limma package and its interface limmaGUI. That's what I do: loading Genepix files background correction (normexp, cutoff=10), setting the GenePix Weightings: unflagged= 1, everything else= 0, within (global loess) and between (Scale) array normalization. To obtain the statistics for differential expression I choose the "least squares" linear model fit and the calculation of Duplicate correlation. Seems to work well, the problem is, that not always all 4 replicates of one mir are "unflagged". It can happen that on the same slide only 2 or 3 replicates are unflagged, or that on one slide all of the 4 replicates are flagged bad, but on the other slides all replicates for the same mir are unflagged. If I understood well, setting the weights only means that the intensities from spots with zero weight do not influence the normalization. How do I exclude the spots with zero weigths from the further calculations of the linear model fit, is there a way in limmaGUI to exlude the bad spots from the toptable list? Thanks a lot in advance. Greetings, Christine Dr. Christine Völlenkle, Ph.D. Research Laboratories-Molecular Cardiology I.R.C.C.S. Policlinico San Donato Via R. Morandi, 30 20097 S. Donato M.se (MI) Italy Phone: +39 02 52774 683 (lab) +39 02 52774 533 (office) Fax: +39 02 52774 666 email: christine.voellenkle@gmail.com [[alternative HTML version deleted]]