This is not strictly a BioConductor question, but a more general one... I hope it's not too off-topic. I am using Limma/LimmaGUI for the analysis of my two-colour expression arrays (cDNA). I have become a "fan" of B values as a tool to rank genes according to their likelihood that they're differentially expressed. Rather than deciding an arbitrary cut-off point (B, P or fold-based) I choose how many genes to work depending on how many I can reasonably deal with, taking into account their ranking according to B. I have just come to a problem, and I was wondering what other more experienced people here think. The story is like this: 1) Using a human 10k array, I did several comparisons between cell lines. I was getting B values of up to 14-15. Everything look good. 2) We got a new array, a 22k one. Hey! more genes! great! This time my experiments were comparisons between treated/untreated in the same cell line. The treatments were various siRNA knockdowns and overexpression of transgenes. The highest B values were around 4... I assumed the much lower values were a reflection of the variation between experiments (every slide was hybridised with a separate treatment)... By Western we could see how our transgene was being expressed at varying levels in different experiments, so it sort of made sense... although the low B values still surprised me a bit. 3) I decided to repeat one of the experiments from 1)... this is a comparison between two straight cell lines, no transfections or any other treatment. Now, while using the 10k array I obtain a max value of B=14.43, when I tried it on my new 22k arrays, the largest B value was a mere 3.69. All hybs were performed in the same way, with the same buffer. RNA extracted and labelled the same way... same scanner... All slides look clean, before and after hyb. Has anybody experienced something like this? I'm beginning to think that the new arrays are not that good... Does anybody know what would be a good way to check that a set of arrays are good enough? I have access to a scanner that can detect DAPI. I stained two old slides and scanned them. The 10k one gave pretty uniform spots. The new 22k one varied wildly (I can email pictures upon request) from spot to spot... but I haven't yet checked whether there's also big differences *between* individual slides. I imagine if the spotted DNA varies in amount a lot, where sometimes you have very little, the ratios could be more variable than we wish, and perhaps that brings the B values down? I really don't know. I have a whole bunch of these arrays and they contain many genes we are interested in... but they'll be pretty useless if I cannot trust the results and makes their interpretation difficult. I would appreciate any help, any pointers as to what I should check to ascertain the quality of any slides we may purchase. Or any comments pointing out to other possible factors I have not considered. Thanks so much! Jose -- Dr. Jose I. de las Heras Email: J.delasHeras at ed.ac.uk The Wellcome Trust Centre for Cell Biology Phone: +44 (0)131 6513374 Institute for Cell & Molecular Biology Fax: +44 (0)131 6507360 Swann Building, Mayfield Road University of Edinburgh Edinburgh EH9 3JR UK