Dear Caryn, I have not evaluated the effect of normalization when there is a blocking factor, and I am not aware of any reference about this. I guess you could normalize within blocks if you have a randomized complete block design and use block as a factor. I would not want to do this with an incomplete block design. I guess as a statistician the issue was obvious to me: Analysis of differential expression compares the variance between treatments to the variance within treatments. Since the effect of normalization is to reduce the variance among the arrays normalized together, and since separate normalizations are likely to introduce variance between the groups, it seems obvious that the net effect of normalizing within treatment group will be to increase the false detections. I guess I do not have a good feel for what needs to be put into the literature. A number of issues that I thought were obvious ended up being worth the effort of experimental follow-up and a paper. People seem to feel that microarray data will behave differently than the data scientists and statisticians have worked with for the past century, but I have seen no evidence that this is so. --Naomi At 04:01 PM 3/1/2006, Caryn M Thompson wrote: >Naomi, > > A copy of your recent posting to the Bioconductor discussion list >was forwarded to me. I haven't seen much in the literature re:the issue >of normalizing within conditions versus across all arrays - could you >point me to some good references? Also, have you done anything to >evaluate the effect of various normalization schemes when a blocking >factor is involved? I've seen a recent argument to suggest that >normalization within blocks is appropriate provided a blocking factor is >included in the model (assuming a linear models approach is taken for >analysis). > >Best regards, >Caryn Thompson > > > >Caryn M. Thompson, PhD >Associate Professor >Director of the Statistical Consulting Center >Department of Bioinformatics and Biostatistics >School of Public Health and Information Sciences >University of Louisville >555 S Floyd St. >Louisville, KY 40292 >Phone: (502) 852-1888 >Fax: (502) 852-3294 Naomi S. Altman 814-865-3791 (voice) Associate Professor Dept. of Statistics 814-863-7114 (fax) Penn State University 814-865-1348 (Statistics) University Park, PA 16802-2111