Identification of artifactual microarray probe signals constantly present in multiple sample types

Abstract

The detection, identification, and quantitation of transcripts have evolved from simple Northern analysis, cDNA cloning, and sequencing to RT-PCR, microarrays, and now digital gene expression using ultra–high-throughput RNA sequencing (RNA-Seq). During the course of our studies we observed that some microarray probes show very high signal intensity values yet are discordant when compared with RNA-Seq. A total of 99 probes from approximately 30,000 were identified as consistently discordant in four human tissues or cell lines. Interestingly, this set of discordant probes appears array-dependent. Among the 99 probes identified, 70 constantly exhibited a high signal in all 713 available samples surveyed using the Illumina HumanHT-12v4 platform. Some were discordant with additional probes that annotated the same genes. Absence of a number of these transcripts was confirmed by quantitative RT-PCR (qRT-PCR). Our findings suggest that one must be cautious, as some array probes do not capture the level of the target.