> > The controls that Affy uses are spiked-in transcripts that are designed > to indicate problems in the hybridization step, which would be > indicative of a 'bad' chip. > > The RNA digestion plot gives a qualitative idea of two things; the > amount of RNA degradation that occurred during the preparation of your > RNA (due presumably to RNAases), and how well the second strand > synthesis went in the preparation of your samples. > > Since these are two completely different things, you cannot compare the > results and ask which one you should believe. You can believe both and > there is no contradiction. > > As an aside, I almost always see a 'significant' slope to the curves in > the RNA digestion plot. I don't necessarily think this is a critical > problem. However, if the curves have completely different slopes, I > start to get worried (not that there is much I can do at that late > stage...). > > Jim > An addition. I would ignore the p-values from the RNA degradation plots. They are not remotely accurate, and will either disappear entirely in future versions of the function, or be calculated very differently. The slope has a much lower coefficient of variation than the best of the individual housekeeping genes. Once experience has shown us which slope values are cause for concern in a given chip type, the slope may be a more sensitive indicator of problems than housekeeping genes. That experience will be essential though for correct interpretation. Leslie Cope