Totally agree with Ramon. The use of a reference sample is a very good practice when you want to compare more than 3 different samples: there are not so many missing values, every pairwise comparison is straight forward and the different properties of the dyes are not a problem any longer. However, the problem is to find this reference. For a time course experiment, we tried using a pool of RNA extracted at the different time points. In theory, the idea was good,specially because we would then obtain a signal in the reference channel for all the interesting genes, those lightening up at any time point, but in practice the number of missing values didn't improve with respect to previous experiments where we used RNA extracted at t0 as reference. Has anyone used a pool of RNA at different time points? For prokaryotes we used gDNA as reference and it worked pretty well...(see Taalat et al. for reference) About the dye swap when we need to balance the different properties of the dyes I always wonder why to use it is such a problem. Unfortunately, for two-color microarrays we usually need one technical repeat to validate the results. Since we have to do it, anyway, why not to swap the dyes and have some extra information that might be also useful for the normalization of the data? Best regards, Fatima -----Original Message----- From: bioconductor-bounces@stat.math.ethz.ch [mailto:bioconductor-bounces@stat.math.ethz.ch] On Behalf Of Ramon Diaz-Uriarte Sent: Friday, October 24, 2003 11:05 AM To: rossini@u.washington.edu; A.J. Rossini; Isaac Mehl; bioconductor Subject: Re: [BioC] dye swap vs. control channel > I think it boils down to whether you are interested in a direct > head-to-head comparison (then dye swapping is essential), or in more > general, less direct statements about groups of samples, with the > possibility of generalizing past the experiment (then choice of > reference sample is critical). Along these linese, if the researcher is certain that she is interested in comparing two (and just these two) groups, then I prefer a dye-swap design. If the question is not as well specified, if more groups could be of interest in the future ---recall connected design issues---, or if they want to try to find predictors (say, discrminant analysis) or, (in spite of admonitions against it) use clustering, I prefer to use a standard (a pool --below), always in the same channel. I think with this approach we tend to view the standard or reference as something we need to live with with cDNA arrays, not something we really care about or wish we had to use ---I guess we wish we had affy arrays. > > in, given the associated costs. Right now, if you have a good > reference or reference prototype for your situation, and can afford > biological replications, I'd probably do reference comparisons. When a reference is used, and seen as a necessary experimental evil, I often want it to be as representative as possible, but at the same time as little variable as possible (i.e., for the reference use aliquots from the pool so that variation from pool to pool sample is nearly inexistent). This is something we have done: form the pool using samples from, say, 30 normal subjects; then, "validate" the pool by using another set of normal samples (not used in the construction of the pool); if the pool is OK, then we should see no genes differentially expressed. (The size of the set of normal samples for "validation" is choosen so that it is at least a little bit larger than the sample size of the experimental conditions). Best, Ram?n > > So all of a sudden, we've moved beyond statistical design, to > cost-function based decision theory and risk analysis. > > Sorry, no easy answers this morning. > > I'd be interested in hearing others opinions on the topic as well. > > best, > -tony > > Isaac Mehl <imehl@ucsd.edu> writes: > > this design brings up a question i always have. is it "better" to do > > all experiments in one channel (Cy5) and compare every sample to a > > standard (cy3)? this way you can use less arrays or do more biological > > replicates. IMHO getting repeated measurements of biological variation > > is more important than dye swap. > > > > very interested to hear what people think about this topic since it is > > integral to experimental design. > > > > -isaac > > > > > > > > DIF1,2 and 3 are different but similar drugs............... > > > >>> Slides 1-6 are treatment 1 (DIF1) Vs No treatment > >>> Slide1 Cy5/Cy3 (DIF1/no treatment) > >>> Slide2 Cy3/Cy5 (DIF1/no treatment) > >>> Slide3 Cy5/Cy3 (DIF1/no treatment) > >>> Slide4 Cy3/Cy5 (DIF1/no treatment) > >>> Slide5 Cy3/Cy5 (DIF1/no treatment) > >>> Slide6 Cy5/Cy3 (DIF1/no treatment) > >>> > >>> Slides 7-12 are treatment 2 (DIF2) Vs No treatment > >>> Slide7 Cy5/Cy3 (DIF2/no treatment) > >>> Slide8 Cy3/Cy5 (DIF2/no treatment) > >>> Slide9 Cy5/Cy3 (DIF2/no treatment) > >>> Slide10 Cy3/Cy5 (DIF2/no treatment) > >>> Slide11 Cy3/Cy5 (DIF2/no treatment) > >>> Slide12 Cy5/Cy3 (DIF2/no treatment) > >>> > >>> Slides 13-18 are treatment 3(DIF3) Vs No treatment > >>> Slide13 Cy5/Cy3 (DIF3/no treatment) > >>> Slide14 Cy3/Cy5 (DIF3/no treatment) > >>> Slide15 Cy5/Cy3 (DIF3/no treatment) > >>> Slide16 Cy3/Cy5 (DIF3/no treatment) > >>> Slide17 Cy3/Cy5 (DIF3/no treatment) > >>> Slide18 Cy5/Cy3 (DIF3/no treatment) > >>> > >>> I'd obviously like to compare across the different treatments DIF1,2 > >>> and 3 > > > > -- > > -isaac mehl > > gene expression lab (gele) > > salk institute > > 10010 n. torrey pines rd. > > la jolla ca. 92037 > > http://genex.salk.edu > > > > _______________________________________________ > > Bioconductor mailing list > > Bioconductor@stat.math.ethz.ch > > https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor -- Ram?n D?az-Uriarte Bioinformatics Unit Centro Nacional de Investigaciones Oncol?gicas (CNIO) (Spanish National Cancer Center) Melchor Fern?ndez Almagro, 3 28029 Madrid (Spain) Fax: +-34-91-224-6972 Phone: +-34-91-224-6900 http://bioinfo.cnio.es/~rdiaz PGP KeyID: 0xE89B3462 (http://bioinfo.cnio.es/~rdiaz/0xE89B3462.asc) _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor