Dear all, I have a question regarding the way to properly design biological replicate and technical replicates. In the projet, we have two groups of sample : R (respond to the treatment), NR (no response). For each groups, there is several replicates (3) that come from different patients (so not strictely biological). Each patient have two sample : one at day one(d1), the second after one week (d7) The hybridization are planned to be performed in dual color, with dye- sap. Here is the target file : Cy3 Cy5 Patient Issu TechnicalReplicat d1 d7 A R 1 d7 d1 A R 1 d1 d7 B R 2 d7 d1 B R 2 d1 d7 C R 3 d7 d1 C R 3 d1 d7 D NR 4 d7 d1 D NR 4 d1 d7 E NR 5 d7 d1 E NR 5 d1 d7 F NR 6 d7 d1 F NR 6 my question is how to properly design the matrix design and the contrast matrix to answer the difference between the two groups ? Which column should I use as biological replicat (so as I can use the duplicateCorrelation function) ? here is how I've started, but I can't figure out how to use duplicateCorrelation with this design design <- cbind( R_D7vsD1 = c(-1,1,-1,1,-1,1, 0,0, 0,0, 0,0), NR_D7vsD1 = c( 0,0,0,0,0,0,-1,1,-1,1,-1,1) ) fit <- lmFit(MAn, design, weights = NULL) cont.mat = makeContrasts(PDvsSD = PD_TvsBL - SD_TvsBL, levels = design) fit2 <- contrasts.fit(fit, cont.mat) fit2 <- eBayes(fit2) Many thanks by advance for any help. -- Guillaume [[alternative HTML version deleted]]

Sorry, I made some mistake in my previous mail, into the contrast matrix (bad copy paste from another project), so here I have corrected them. > I have a question regarding the way to properly design biological replicate and technical replicates. > > In the projet, we have two groups of sample : R (respond to the treatment), NR (no response). > For each groups, there is several replicates (3) that come from different patients (so not strictely biological). > Each patient have two sample : one at day one(d1), the second after one week (d7) > The hybridization are planned to be performed in dual color, with dye-sap. > > Here is the target file : > > Cy3 Cy5 Patient Issu TechnicalReplicat > d1 d7 A R 1 > d7 d1 A R 1 > d1 d7 B R 2 > d7 d1 B R 2 > d1 d7 C R 3 > d7 d1 C R 3 > d1 d7 D NR 4 > d7 d1 D NR 4 > d1 d7 E NR 5 > d7 d1 E NR 5 > d1 d7 F NR 6 > d7 d1 F NR 6 > > > my question is how to properly design the matrix design and the contrast matrix to answer the difference between the two groups ? > Which column should I use as biological replicat (so as I can use the duplicateCorrelation function) ? > > here is how I've started, but I can't figure out how to use duplicateCorrelation with this design > > design <- cbind( > R_D7vsD1 = c(-1,1,-1,1,-1,1, 0,0, 0,0, 0,0), > NR_D7vsD1 = c( 0,0,0,0,0,0,-1,1,-1,1,-1,1) > ) > fit <- lmFit(MAn, design, weights = NULL) > cont.mat = makeContrasts(NRvsR = NR_D7sD1 - R_D7vsD1, levels = design) > fit2 <- contrasts.fit(fit, cont.mat) > fit2 <- eBayes(fit2) > > > > > Many thanks by advance for any help. > > -- > Guillaume > [[alternative HTML version deleted]] > > _______________________________________________ > Bioconductor mailing list > Bioconductor at r-project.org > https://stat.ethz.ch/mailman/listinfo/bioconductor > Search the archives: http://news.gmane.org/gmane.science.biology.informatics.conductor