Dear list members: Hello. I am trying to convert the tiff images of RayBio Cytokine Antibody Array (http://www.raybiotech.com/product.htm) into numbers in order to compare them numerically. The problem I am having now is that how to define the area of a spot. Do any of you have experience with working with protein arrays? Any help is appreciated. Thank you very much for your help in advance. Shizuka

Hi Shizuka: Which software are you using for segmentation of the images? Some software (e.g., ImaGene) provide a way of increasing or decreasing the circle size to cover the area of the spot manually. But manually finding spot area is (1) too time-consuming. (2)visual judgment can be misleading, it is influenced by the levels of brightness, contrast etc. (3) the spots might be irregular in shape, so fitting a circle would be erroneous. It is best to automate the whole process and only check at the end to see if any obvious mistake was done. Spot uses a slightly different algorithm (from ImaGene) and, I think (others might give better opinion) produces more realistic values for the spot intensities. You could download an evaluation copy of Spot(http://spot.cmis.csiro.au/spot/index.php) and try it out. Best wishes, Saroj > Dear list members: > > Hello. I am trying to convert the tiff images of RayBio Cytokine > Antibody Array (http://www.raybiotech.com/product.htm) into numbers in > order to compare them numerically. > The problem I am having now is that how to define the area of a spot. > Do any of you have experience with working with protein arrays? Any > help is appreciated. Thank you very much for your help in advance. > > Shizuka > > _______________________________________________ > 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 >

Dear Uchida, I tried to analyze a similar protein array. You can have a look to: https://stat.ethz.ch/pipermail/bioconductor/2006-June/013311.html What I did was to invert the black and the white image, so that the spots appear in white and the background in black and I analyzed the images as if they were a Cy3 image with the commercial program GenePix. There are another programs to obtain the signal intensity and the background (GridGrinder {free} [http://gridgrinder.sourceforge.net/], SpotFinder {free}[http://www.tm4.org/spotfinder.html], Spot {free 1 month}[http://spot.cmis.csiro.au/spot/index.php], ). About Spot, which has been recommended, I didn?t find how to manage with ?one channel? arrays, as the protein arrays (perhaps I am wrong). There is also an R package (apart from Spot), which is Spotsegmentation, although I don?t know how to manage it, and there was some mails asking about it without any answer. I recommend you that you have a look to: http://ihome.cuhk.edu.hk/~b400559/arraysoft_image.html Although it?s a little bit out of date (2004), it?s quite useful. HTH, Jordi Altirriba PhD student Hospital Clinic, Barcelona, Spain >Hello. I am trying to convert the tiff images of RayBio Cytokine Antibody >Array (http://www.raybiotech.com/product.htm) into numbers in order to >compare them numerically. >The problem I am having now is that how to define the area of a spot. Do >any of you have experience with working with protein arrays? Any help is >appreciated. Thank you very much for your help in advance.