The reference design is a practical and popular choice for microarray studies using two-color platforms. In the reference design, the reference RNA uses half of all array resources, leading investigators to ask: What is the best reference RNA? We propose a novel method for evaluating reference RNAs and present the results of an experiment that was specially designed to evaluate three common choices of reference RNA. We found no compelling evidence in favor of any particular reference. In particular, a commercial reference showed no advantage in our data. Our experimental design also enabled a new way to test the effectiveness of pre-processing methods for two-color arrays. Our results favor using an intensity-normalization and foregoing background-subtraction. Finally, we evaluate the sensitivity and specificity of data quality filters, and propose a new filter that can be applied to any experimental design and does not rely on replicate hybridizations.
Bioinformatics | Computational Biology | Microarrays
Kerr, Kathleen F.; Serikawa, Kyle A.; Wei, Caimiao; Peters, Mette A.; and Bumgarner, Roger E., "What Is the Best Reference RNA? And Other Questions Regarding the Design and Analysis of Two-Color Microarray Experiments" (April 2007). UW Biostatistics Working Paper Series. Working Paper 305.