I need analyse the macroarrays. Im my case, I dont have Cy3 and Cy5. I have 2 nylon membranes for each replicaties: one control and one treated. In my experiments, I have 3 replicaties. The softwere image analyze have 1 out put files for each 2 nylon membrane with 3 colunms for control and 3 colunms for treated, like this (Ctrl=control, Data=treated): Ctrl Ctrl Ctrl Data Data Data VOL Bkgd sVOL VOL Bkgd sVOL A - 1 : 1 (1) 14.3 13.7 0.5 481.0 420.3 60.7 My question: - In the Targets file have four essential columns (I read it in the limmaGUI man in web site). The colunms Cy3 and Cy5 are essential for limmaGUI? Thanks Marcelo ---

"Marcelo Luiz de Laia" <mlaia@fcav.unesp.br> writes: > I need analyse the macroarrays. > > Im my case, I dont have Cy3 and Cy5. I have 2 nylon membranes for each > replicaties: one control and one treated. In my experiments, I have 3 > replicaties. The softwere image analyze have 1 out put files for each 2 > nylon membrane with 3 colunms for control and 3 colunms for treated, like > this (Ctrl=control, Data=treated): > > Ctrl Ctrl Ctrl Data Data Data > VOL Bkgd sVOL VOL Bkgd sVOL > A - 1 : 1 (1) 14.3 13.7 0.5 481.0 420.3 60.7 > > My question: > - In the Targets file have four essential columns (I read it in the limmaGUI > man in web site). The colunms Cy3 and Cy5 are essential for limmaGUI? Gordon can probably answer better, but you might need to set either Cy5 or Cy3 == 1 and just treat the single channel as a ratio. It's one reasonable first step. However, for radioactive cDNA arrays (nylon filter membranes), what I usually do is to use an exprSet. You don't state which filters you are using, but the basic idea would be to: 1. decide if you want to background correct, or not (filters are funny about this, compared to glass spotted's). If the machine gives you "expression values", you might consider using them. (this is NOT a promise, just that if you know what happened to provide expression values, you might want to use them). You might have to adjust for imaging times, etc, to get comparable data between chips (adjusting norms). 2. Once you've got these expression values, between-chip normalize via VSN. 3. Now use esApply to compute whatever it is you want to compute for determining differential expression, or plug into heatmap if you want a first pass at 2-way clustering. This works reasonably well with the filters and lab I'm used to working with. Your mileage may vary wildly depending on the labs (and filters) that you are working with. best, -tony -- rossini@u.washington.edu http://www.analytics.washington.edu/ Biomedical and Health Informatics University of Washington Biostatistics, SCHARP/HVTN Fred Hutchinson Cancer Research Center UW (Tu/Th/F): 206-616-7630 FAX=206-543-3461 | Voicemail is unreliable FHCRC (M/W): 206-667-7025 FAX=206-667-4812 | use Email CONFIDENTIALITY NOTICE: This e-mail message and any attachme...{{dropped}}

Marcelo, The current version of limmaGUI which you have is only designed for two-color cDNA arrays, and yes it does assume that you have at least these essential columns in the Targets input file: SlideNumber, FileName, Cy3 and Cy5 (for Spot, GenePix image analysis files. May also work for QuantArray but haven't really tested these yet.) For ImaGene image analysis files, the column headings are the same except FileName is replaced by two columns, FileNameCy3 and FileNameCy5. Which image analysis software are you using? I am working on an affy version of limmaGUI. I have a rough prototype almost working for the estrogen example on the Bioconductor site. But I don't think this will help you... It sounds like your arrays are cDNA not affy, but they are just different to the arrays I've seen in that they don't have two dyes. Maybe you could try something like this: SlideNumber Cy3 Cy5 1 Ref Control 2 Ref Treated Then when you look at M values for one slide (not between slides), the M value is log2(RedIntensity/GreenIntensity) or log2(Cy5Intensity/Cy3Intensity), so the M values you get for slide 1 in the example above would be log2(Control/Ref) Does that make sense? Maybe I'm still not understanding your experiment very well. Regards, James ---------------------------------------------------------------------- ---- James Wettenhall Tel: (+61 3) 9345 2629 Division of Genetics and Bioinformatics Fax: (+61 3) 9347 0852 The Walter & Eliza Hall Institute E-mail: wettenhall@wehi.edu.au of Medical Research, Mobile: (+61 / 0 ) 438 527 921 1G Royal Parade, Parkville, Vic 3050, Australia http://www.wehi.edu.au