I have some questions about Affymetrix Poly-A Controls. The Affymetrix Data Analysis Fundamentals manual states that "Poly-A RNA controls can be used to monitor the entire target labeling process....All of the Poly-A controls should be called "Present" with increasing Signal values in the order of lys,phe,thr,dap." I have some array data for which the Poly-A controls (lys, phe, thr, dap) are called Absent on many arrays. In addition, the signal values consistently follow the order lys<phe<dap<thr across="" arrays.="" to="" look="" at="" this,="" i="" took="" the="" average="" of="" all="" probe="" sets="" for="" each="" of="" the="" four="" controls="" and="" plotted="" these="" values="" by="" array.="" i="" should="" note="" that="" the="" hybridization="" controls="" (biob,="" bioc,="" biod="" and="" cre)="" look="" good.="" in="" addition,="" i="" verified="" that="" the="" genechip="" poly-a="" rna="" control="" kit="" was="" used.="" does="" anyone="" have="" experience="" with="" experiments="" where="" the="" poly-a="" controls="" did="" not="" perform="" as="" expected?="" what="" does="" this="" tell="" me="" about="" the="" data="" and="" the="" experimental="" protocol?="" should="" this="" have="" been="" checked="" by="" the="" core="" lab="" technician?="" any="" insight="" into="" this="" problem="" would="" be="" greatly="" appreciated!="" ann="" <="" div="">

Hi Ann, Ann Hess wrote: > I have some questions about Affymetrix Poly-A Controls. > > The Affymetrix Data Analysis Fundamentals manual states that "Poly-A > RNA controls can be used to monitor the entire target labeling > process....All of the Poly-A controls should be called "Present" with > increasing Signal values in the order of lys,phe,thr,dap." > > I have some array data for which the Poly-A controls (lys, phe, thr, dap) > are called Absent on many arrays. In addition, the signal values > consistently follow the order lys<phe<dap<thr across="" arrays.="" to="" look="" at=""> this, I took the average of all probe sets for each of the four controls > and plotted these values by array. I should note that the Hybridization > Controls (bioB, bioC, bioD and cre) look good. In addition, I verified > that the GeneChip Poly-A RNA Control Kit was used. > > Does anyone have experience with experiments where the Poly-A controls did > not perform as expected? What does this tell me about the data and the > experimental protocol? Should this have been checked by the core lab > technician? I don't use mas5 numbers, and don't much believe in the utility of the P/M/A calls, so cannot comment on whether or not these controls are usually called present. However, when I have looked at these controls, I pretty much always see the same pattern that you mention, although the dap control expression value can vary from dap<lys<phe<thr to="" lys<phe<dap<thr.="" i="" am="" not="" so="" sure="" these="" controls="" are="" that="" useful,="" and="" i="" would="" personally="" look="" at="" other="" measures="" of="" quality="" to="" assure="" myself="" that="" the="" data="" were="" of="" reasonable="" quality.="" best,="" jim=""> > Any insight into this problem would be greatly appreciated! > > Ann > > _______________________________________________ > 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 -- James W. MacDonald, M.S. Biostatistician Affymetrix and cDNA Microarray Core University of Michigan Cancer Center 1500 E. Medical Center Drive 7410 CCGC Ann Arbor MI 48109 734-647-5623 ********************************************************** Electronic Mail is not secure, may not be read every day, and should not be used for urgent or sensitive issues.