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The scaling factor won't take care of all of the problems (It will actually cause some problems). We have successfully prepared 200ul hybridization cocktails using 10ug or less of cRNA. Use 15ul if you have 15ug cRNA, 10 if you have 10 or less. This helps to compensate. See McClintick, et al. BMC Genomics 2003 for comparison of arrays using different amounts of cRNA for hybridization. If you have much less than 10ug, you will probably still have some problems. I wouldn't go any lower than 7ug. Check the % present and scaling factor to see if they are within reason. (+/- 2Std. deviations). Contact me directly if want to discuss this further. Jeanette McClintick, PhD Indiana University School of Medicine Center for Medical Genomics Indianapolis, IN 317-274-8488 jnmcclin at iupui.edu -----Original Message----- From: bioconductor-bounces@stat.math.ethz.ch [mailto:bioconductor-bounces at stat.math.ethz.ch] On Behalf Of IAIN GALLAGHER Sent: Monday, January 14, 2008 10:43 AM To: bioconductor at stat.math.ethz.ch Subject: [BioC] OT: Question about affymetrix array analysis and cRNA amount. Hi. Sorry about the off topic (not bioconductor) question but with so many statisticians and microarray users on the list I hoped it would be a good place to post. I am new to microarray work and we have clinical samples we would like to run on Affymetrix chips. The RNA looks good by various analysis but the amount of cRNA from many of the samples is suboptimal. My question is this: can we compensate for using different amounts of cRNA at the analysis stage e.g. using a scaling factor or other means? For instance if one chip ran with 15ug cRNA and another with 7ug can this be accomodated sensibly or would the comparison of these two chips be essentially meaningless? Thank you for any advice. Iain [[alternative HTML version deleted]] _______________________________________________ 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