Hi, I am analyzing a small microarray experiment with 5 RAE230_2 chips with material from two strains of rat. I loaded in the data using ReadAffy and then run the rma function to normalize the data and produce the expression values. In 486 (out of 31099) probesets, the expression values are identical over all 5 chips and in two others there was no variability within strain although there was across strains. In either of these cases, I get zero variability for the mean difference. I have examined the original probe level data as read from the CEL files and there is variability across the chips in both the PM and MM measurements so my conclusion is that this is an effect of the rma or normalization steps. Has anyone else witnessed this effect? What is the preferred way to deal with this lack of variability? Thanks for any help that you can provide. Angelo -- ------------------------------------------------------------------ | Angelo J. Canty Email: cantya@mcmaster.ca | | Mathematics and Statistics Phone: (905) 525-9140 x 27079 | | McMaster University Fax : (905) 522-0935 | | 1280 Main St. W. | | Hamilton ON L8S 4K1 |

This is an artifact of the medianpolish algorithm. If you are using 3 or 5 chips you will get some expression values that are identical for all chips (much worse with 3 chips). You can see why this is if you take the quantile normalized pm values from one of these genes and run medpolish() on them (note here that they have to be normalized!). What happens is that the overall median is subtracted out, then the row median, but at this point the column median for all columns is zero. Since the expression values are the overall median + column median, the expression values are identical for all chips. I don't think there is anything you could/should do with these data. Long story short, these genes are likely not changing expression so they are probably not interesting anyway. If you are having problems with zeros in your denominators, you could simply remove the 486 genes and then proceed. HTH, Jim James W. MacDonald Affymetrix and cDNA Microarray Core University of Michigan Cancer Center 1500 E. Medical Center Drive 7410 CCGC Ann Arbor MI 48109 734-647-5623 >>> Angelo Canty <canty@math.mcmaster.ca> 06/07/04 02:06PM >>> Hi, I am analyzing a small microarray experiment with 5 RAE230_2 chips with material from two strains of rat. I loaded in the data using ReadAffy and then run the rma function to normalize the data and produce the expression values. In 486 (out of 31099) probesets, the expression values are identical over all 5 chips and in two others there was no variability within strain although there was across strains. In either of these cases, I get zero variability for the mean difference. I have examined the original probe level data as read from the CEL files and there is variability across the chips in both the PM and MM measurements so my conclusion is that this is an effect of the rma or normalization steps. Has anyone else witnessed this effect? What is the preferred way to deal with this lack of variability? Thanks for any help that you can provide. Angelo -- ------------------------------------------------------------------ | Angelo J. Canty Email: cantya@mcmaster.ca | | Mathematics and Statistics Phone: (905) 525-9140 x 27079 | | McMaster University Fax : (905) 522-0935 | | 1280 Main St. W. | | Hamilton ON L8S 4K1 | _______________________________________________ Bioconductor mailing list Bioconductor@stat.math.ethz.ch https://www.stat.math.ethz.ch/mailman/listinfo/bioconductor