Dear all, I?ve got the following microarray experiment design-related problem when using Limma: In our lab we want to perform some microarray hybridizations to identify genes whose expression is altered by a certain treatment over a time. We want to do cohybridizations of RNA from treated vs untreated cells at corresponding time points to spotted cDNA arrays. This time series should be performed in triplicate, including three biological replicated and a dye-swap Tx=treated, Ux=untreated cells at time point x * T2 ? U2 * T6 ? U6 * T12 ? U12 My problem is the following: Is it statistically valid to estimate the interaction Time:Treatment using such an unconnected design? Using the following design matrix T2vsU2 T6vsU6 T12vsU12 [1,] 1 0 0 [2,] 1 0 0 [3,] -1 0 0 [4,] 0 1 0 [5,] 0 1 0 [6,] 0 -1 0 [7,] 0 0 1 [8,] 0 0 1 [9,] 0 0 -1 and contrast matrix? Contrasts Levels Interaction_6_and_2_hours Interaction_12_and_6_hours T2vsU2 -1 0 T6vsU6 1 -1 T12vsU12 0 1 the script can be executed without error message using R (vers. 2.4.0) and Limma (2.9.8), while the same kind of analysis in LimmaGUI (1.10.0) produces the following error message?: ?For this experiment, all possible RNA comparisons can be obtained from one parameterization. This is either because there are only 2 RNA types, or there is an unconnected experimental design and there only 2 RNA types within each sub-experiment.? A former post from 2006-10-27 by Richard Friedman was already related to the same kind of error message in LimmaGUI. What I wanted to know is wether this message is specific to LimmaGUI and its implementation as compared to Limma or does it point out that this design is not appropriate for this kind of analysis as in Limma no error message is displayed. I would appreciate any comment or suggestion. Best regards Serge Serge Eifes Laboratoire de Biologie Moleculaire et Cellulaire du Cancer (LBMCC) Hopital Kirchberg 9,rue Edward steichen L-2540 LUXEMBOURG Phone:+ 352 2468-4046 ?Fax : + 352 2468-4060

This is a complete factorial "treatment design" with 2 factors, time and treatment. You have set it up correctly to obtain the interaction effects. If you use single channel analysis, set this up as factorial design with array as block. Regards, Naomi At 09:27 AM 2/8/2007, Serge Eifes wrote: >Dear all, >I?ve got the following microarray experiment design-related problem >when using Limma: In our lab we want to perform some microarray >hybridizations to identify genes whose expression is altered by a >certain treatment over a time. We want to do cohybridizations of RNA >from treated vs untreated cells at corresponding time points to >spotted cDNA arrays. This time series should be performed in >triplicate, including three biological replicated and a dye-swap > >Tx=treated, Ux=untreated cells at time point x >* T2 ? U2 >* T6 ? U6 >* T12 ? U12 > >My problem is the following: Is it statistically valid to >estimate the interaction Time:Treatment using such an unconnected >design? > >Using the following design matrix > > T2vsU2 T6vsU6 T12vsU12 > [1,] 1 0 0 > [2,] 1 0 0 > [3,] -1 0 0 > [4,] 0 1 0 > [5,] 0 1 0 > [6,] 0 -1 0 > [7,] 0 0 1 > [8,] 0 0 1 > [9,] 0 0 -1 > >and contrast matrix > > Contrasts >Levels Interaction_6_and_2_hours Interaction_12_and_6_hours > T2vsU2 -1 0 > T6vsU6 1 -1 > T12vsU12 0 1 > >the script can be executed without error message using R (vers. 2.4.0) >and Limma (2.9.8), while the same kind of analysis in LimmaGUI (1.10.0) >produces the following error message : > >?For this experiment, all possible RNA comparisons can be obtained from >one parameterization. >This is either because there are only 2 RNA types, or there is an >unconnected experimental design and there only 2 RNA types within each >sub-experiment.? > >A former post from 2006-10-27 by Richard Friedman was already related >to the same kind of error message in LimmaGUI. What I wanted to know >is wether this message is specific to LimmaGUI and its implementation >as compared to Limma or does it point out that this design is not >appropriate for this kind of analysis as in Limma no error message is >displayed. > >I would appreciate any comment or suggestion. > >Best regards >Serge > > >Serge Eifes >Laboratoire de Biologie Moleculaire et Cellulaire du Cancer (LBMCC) >Hopital Kirchberg >9,rue Edward steichen >L-2540 LUXEMBOURG >Phone:+ 352 2468-4046 Fax : + 352 2468-4060 > >_______________________________________________ >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 Naomi S. Altman 814-865-3791 (voice) Associate Professor Dept. of Statistics 814-863-7114 (fax) Penn State University 814-865-1348 (Statistics) University Park, PA 16802-2111