
We used Mas5 generated data to do analysis (floor the data, p value cut off, and further filter by fold change) and found out that RTPCR was able to validate the differentially regulated genes 80-85% of the time (p<0.05 and fold>2) based on RTPCR of more than 100 genes.  I have been using RMA rencently and am curious to know whether any one has done extensive RTPCR validateion of the RMA results.

Secondly,  how well does RMA handle outlier chip?  I recently ran RMA using 16 chips and compared the results with 16 chips + one outlier chip.  To my surprise, the results are vastly different.

Thirdly, since the assumption for RMA is that chip-chip variance is usually bigger than true variance within a certain gene, I am not sure whether I need to run a single, or multiple RMA for a time course experiment that I am expecting to see significant difference.  For example, I am looking at ligand effect on certain cells at 2, 4, and 8 hours.  Not sure whether it's better to group the ligand treated experiments together (12 chips) and the controls together (16 chips) and combine them later, or group them together to do RMA.   The results, I mean, the differentially regulated genes pulled out by t test are not that similar.

Finally, a couple of people I know that work in different institutions commented that Mas5 works better on human diseased samples (including PBMC) when the inter- and intragroup variance are significant; while RMA works better with tissure cultured samples.

Any comments are well appreciated.

thanks, yihong 


		
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