Hello Heidi, thanks for you reply, problem solved! no error anymore. I do not have replicates of each miRNA in the card, so after setting ndups=2 I am getting no error and a result that makes sense. I had a typo in my previous message I have to treatments but the replicates is 2x3 and not 2x2, but that is ok for the test right? It would be better to have a balanced design, but that is what I have available. Thanks again, with kind regards, Andreia On Thu, May 20, 2010 at 10:38 PM, Heidi Dvinge <heidi@ebi.ac.uk> wrote: > Hello Andreia, > > > Dear all, > > > > I am using HTqPCR to analyse different expression of miRNAs, where I am > > comparing two treatments, 1 with 2 biological replicates and the second > > with > > 2 biological replicates > > > Okay, so you have two samples with two replicates each. > > > I have followed the vignete, read the files, made raw.cat and filtered > out > > IQR<1.5 and category= undetermined and type=endogenous > > and everything went well until the fit test: > > > > qDE.limma<-limmaCtData(iqr.filt, design=design, contrasts=contrasts, > > ndups=2, spacing=1) > > Error in dim(M) <- c(spacing, ndups, ngroups, nslides) : > > dims [product 396] do not match the length of object [402] > > > What's the dimensions of your data, i.e. number of genes on each card? > show(iqr.filt) What does traceback() say? > > Using ndups=2 and spacing=1 means that there's a replicate of each gene, > and they're located right next to each other. Is this the case you your > array? > > > can someone tell me what this means? it still performed the test but I > > would > > like to clear out this. > > > > The other weird thing is the result of the test: > > > > genes feature.pos t.test p.value adj.p.value ddCt > > meanTarget meanCalibrator categoryTarget > > 39 miR-122a H7;H8 6.7414852 6.141932e-06 0.0002456773 > 14.8264063 > > 34.833906 20.007500 Undetermined > > 1 miR-18a A1;A2 5.5408137 5.366491e-05 0.0010732983 > 13.5365104 > > 30.431094 16.894583 Undetermined > > 10 miR-368 B11;B12 -4.7841570 2.320152e-04 0.0030935366 > -23.1045833 > > 7.119063 30.223646 Undetermined > > > > the feature.pos is different in both treatments but it is not, I have > > checked and re-checked the files and miR-18a is in pos. A1 in all cards. > > > This is because of ndups=2, spacing=1 that you've specified when you did > the test. It then says that miR-18a is in position A1, but since it should > be replicated and the replicates are next to each other, it must also be > in A2. Ditto for H7;H8 and B11:B12. > > If your spots on the plate are not replicated, you need to set ndups=1. If > they're replicated but not next to each other, you either need to adjust > the spacing parameter accordingly, or sort your file so they are. > ?limmaCtdata gives an example of how to reorder data to get the genes in > consecutive rows. > > When you filter your data, have you then got the same number of replicates > of each spot afterward? What does table(featureNames(iqr.filt)) say? > > HTH > \Heidi > > > Thanks for your help. > > With kind regards > > > > Andreia > > -- > > -------------------------------------------- > > Andreia J. Amaral > > Unidade de Imunologia Clínica > > Instituto de Medicina Molecular > > Universidade de Lisboa > > email: andreiaamaral@fm.ul.pt > > andreia.fonseca@gmail.com > > > > > -- -------------------------------------------- Andreia J. Amaral Unidade de Imunologia Clínica Instituto de Medicina Molecular Universidade de Lisboa email: andreiaamaral@fm.ul.pt andreia.fonseca@gmail.com [[alternative HTML version deleted]]