Hi thanks for the advice Robert, I'm new to this. Anyway here's my list of commands: library(limma) Cy3 <- "F532 Median - B532" Cy3b <- "B532 Mean" targets <- readTargets("targets.csv") RG <- read.maimages( targets$FileName, source="genepix",columns=list(R=Cy3,G=Cy3, Rb=Cy3b, Gb=Cy3b)) # remove the extraneous values red channel values RG$R <- NULL RG$Rb <- NULL pData <- data.frame(population = c('a', 'a', 'a', 'a', 'b', 'b', 'b')) rownames(pData) <- RG$targets$FileName design <- model.matrix(~factor(pData$population)) library('vsn') mat <- vsnMatrix(RG$G) rownames(mat at hx) <- RG$genes$Name mat at hx <- mat at hx[order(rownames(mat at hx)), ] corfit <- duplicateCorrelation(mat, design, ndups=4) fit <- lmFit(mat, design, ndups=4, correlation=corfit$consensus) ebayes <- eBayes(fit) topTable(ebayes, coef = 2, adjust = "BH", n = 399, lfc=1.5) The full script with a whole load of comments included and other stuff included can be viewed here: http://article.gmane.org/gmane.science.biology.informatics.conductor/1 8032/match=miRNA And here's my output of SessionInfo(): R version 2.6.2 (2008-02-08) i686-pc-linux-gnu locale: LC_CTYPE=en_IE.UTF-8;LC_NUMERIC=C;LC_TIME=en_IE.UTF-8;LC_COLLATE=en_IE .UTF-8;LC_MONETARY=en_IE.UTF-8;LC_MESSAGES=en_IE.UTF-8;LC_PAPER=en_IE. UTF-8;LC_NAME=C;LC_ADDRESS=C;LC_TELEPHONE=C;LC_MEASUREMENT=en_IE.UTF-8 ;LC_IDENTIFICATION=C attached base packages: [1] tools stats graphics grDevices utils datasets methods [8] base other attached packages: [1] statmod_1.3.6 vsn_3.2.1 affy_1.16.0 [4] preprocessCore_1.0.0 affyio_1.6.1 Biobase_1.16.3 [7] limma_2.12.0 loaded via a namespace (and not attached): [1] grid_2.6.2 lattice_0.17-6 rcompgen_0.1-17 Hope thats better, Paul Geeleher Department of Mathematics National University of Ireland Galway Ireland On Thu, Jun 5, 2008 at 2:41 PM, Robert Gentleman <rgentlem at="" fhcrc.org=""> wrote: > Hi Paul, > > Please check the posting guide and provide us with the information > requested there (like sessionInfo and the commands you ran). And I typically > don't give any advice (excpet to follow the posting guide) to people who > don't use signatures that identify them. > > Robrt > > > Paul Geeleher wrote: >> >> Hi, >> >> This is the first time I've ever analyzed a microarray experiment >> using Limma (or anything else for that matter) and I was hoping that >> somebody could look at my results and tell me if they look normal. >> >> The experiment is measuring differential expression between miRNAs of >> HER2+ and HER2- breast cancer tissue. There are 3 HER2+ arrays and 4 >> HER2- arrays and each of the 399 miRNAs is replicated 4 times in each >> array. >> >> TopTable() reveals the following miRNAs with a fold change above 1.5, >> which I thought was a reasonable cutoff: >> >> ID logFC t P.Value adj.P.Val >> B >> 273 hsa-miR-451 -4.645060 -8.226854 4.510441e-09 9.246404e-07 >> 10.8484797 >> 128 hsa-miR-205 3.551495 7.574564 2.370061e-08 3.239083e-06 >> 9.2222865 >> 13 hsa-miR-101 -2.310652 -6.569497 3.374177e-07 2.567796e-05 >> 6.6146751 >> 282 hsa-miR-486 -2.686910 -6.542808 3.626060e-07 2.567796e-05 >> 6.5439656 >> 55 hsa-miR-144 -2.890719 -5.889594 2.152998e-06 1.261042e-04 >> 4.7952480 >> 387 mmu-miR-463 -2.609257 -5.764143 3.042120e-06 1.559086e-04 >> 4.4561920 >> 388 mmu-miR-464 -2.080402 -5.696976 3.662006e-06 1.668247e-04 >> 4.2743601 >> 151 hsa-miR-223 -1.722956 -5.637290 4.318942e-06 1.770766e-04 >> 4.1126276 >> 51 hsa-miR-142-3p -3.262824 -5.397809 8.386312e-06 3.125807e-04 >> 3.4626378 >> 14 hsa-miR-101_MM1 -1.922710 -5.224075 1.358743e-05 4.175776e-04 >> 2.9905370 >> 159 hsa-miR-26b_MM2 -2.221853 -5.206724 1.425875e-05 4.175776e-04 >> 2.9433849 >> 236 hsa-miR-376a_MM1 -1.633555 -4.653220 6.637043e-05 1.700742e-03 >> 1.4433277 >> 266 hsa-miR-432* 1.512622 4.627293 7.131510e-05 1.719952e-03 >> 1.3734422 >> 168 hsa-miR-29b -1.954087 -4.198854 2.323860e-04 4.763912e-03 >> 0.2280262 >> 31 hsa-miR-126*_MM2 -1.537988 -3.209957 3.233842e-03 5.099520e-02 >> -2.2888897 >> 52 hsa-miR-142-5p -1.881192 -2.831493 8.332384e-03 9.002153e-02 >> -3.1731794 >> >> >> Another person is sanity testing this data using GeneSpring and they >> are getting much higher p-values compared to mine. They are also >> taking the step of excluding quite a few of the miRNAs from the >> experiment based on their standard deviation across the arrays of each >> group. Should I be doing this also or is this taken into account by >> the eBayes() function or lmFit()? >> >> If you are interested the script I wrote to do the analysis is here: >> >> http://article.gmane.org/gmane.science.biology.informatics.conducto r/18032/match=miRNA >> >> Thanks for any advice, >> >> -Paul. >> >> _______________________________________________ >> Bioconductor mailing list >> Bioconductor at stat.math.ethz.ch >> https://stat.ethz.ch/mailman/listinfo/bioconductor >> Search the archives: >> http://news.gmane.org/gmane.science.biology.informatics.conductor >> > > -- > Robert Gentleman, PhD > Program in Computational Biology > Division of Public Health Sciences > Fred Hutchinson Cancer Research Center > 1100 Fairview Ave. N, M2-B876 > PO Box 19024 > Seattle, Washington 98109-1024 > 206-667-7700 > rgentlem at fhcrc.org >