Probe set algorithms: is there a rational best bet?

Current Affymetrix microarrays [1] use chemical synthesis of 25 mer oligonucleotides in 8 μm2 features on glass slides, resulting in a density of approximately 1 million oligonucleotides per 1.3 cm2 microarray. The impressive density of oligonucleotides provides adequate space on the chip ("real estate") for use of multiple probes per mRNA transcript. Thus, each transcript is queried by a probe set (typically 22 oligonucleotides on current arrays). The assessment of the amount of a specific mRNA in a solution from a tissue or cell is determined by the amount of hybridization to the different oligo features for that mRNA. While "hybridization" is a highly sensitive and specific assay, it is not perfectly sensitive or perfectly specific. Thus, it is critical to provide measures of non-specific hybridization and probe binding (background), and one method is to print mismatch probes, where a single base mutation in the center of each 25 mer probe should disrupt most "specific" hybridization. Current expression profiling microarrays use 11 perfect match and mismatch probe pairs for each probe set, although there may be many probe sets per transcript unit.The multiple measurements of hybridization for each mRNA allow a more robust measure of expression, as "signal" is derived from many assessments. However, the multiple measures has led to considerable debate with regards to the best method of integrating the 11 perfect match and 11 paired mismatch hybridization intensities into an ensembled "signal" for each gene. There are many calculations intrinsic to the probe set algorithms that derive gene expression signal, including varied penalties for the mismatch signal, different intra-probe set normalizations, and normalizations between microarrays. Recent publications have reviewed the specific calculations used by different probe set algorithms [2,3]. To illustrate the effects of choice of probe set algorithm on data interpretation, a 27 time point series data was analyzed