1. Dilution/Mixture Study Bill Craven, GeneLogic, Inc. Motivated by a desire for a data set to be used as a baseline to characterize analysis and normalization methods We wanted to examine: –Post-hybridization variability (i.e. scanner effects) –Effects on differential expression estimates compared to known dilution and/or mixing ratios

2. Design Dilutions and mixtures taken out of master IVT preparations pooled for two samples: human liver (sample A) and a central nervous system cell line (sample B). These were chosen to maximize independence of expression. Each dilution or mixture had five replicate HGU95A assays run on five scanners. The concentration of each material is characterized by the number of ug of labeled cRNA in the individual hybridization solutions. All examples had Genelogic’s standard bacterial spikeins added at their nominal concentrations to give an absolute control.

3. Design summary:

4. Unnormalized, MAS 5.0

5. Standard Curve Normalized, MAS 5.0

6. Adjusted Trimmed Mean Normalization, MAS 5.0

7. Initial Observations A fundamental assumption behind our dilution study was that the assays are independent of total cRNA in the hybridization mixture. A quick examination of the raw intensities of the spikeins tells us this isn’t the case; there appears to be a chemical saturation effect whereby at lower total cRNA concentrations, the spike-in signals are higher (this is confirmed at the probe level). Furthermore, this effect introduces significant non- linearities to the expression in the bulk of the sample observed.

8. Median spike-in Intensities vs total RNA Total RNA,  g 11 Randomly selected probe set medians vs total RNA Total RNA,  g MAS 5 Intensity

9. Nominal Ratio = log2(1.25/20) Spike-in Nominal Ratio Spike-ins Log2 Intensities, M vs A: Liver 1.25 ug – Liver 20 ug, scanner 1

10. Looking Forward Further Work: –Complete analysis of dilutions to characterize and normalize out the observed non-linearities –Check observed fold changes against nominal ratios for various methods of analysis –Finally, check for unexpected effects in the mixture cases, indicating some background competition Acknowledgments: –Terry Speed, Harry Zuzan, Francois Collin for improvements in the design –Uwe Scherf, Yasmin Beazer-Barclay for making the wet-lab part happen!