Affymetrix probes containing runs of contiguous guanines are not gene-specific

Abstract

High Density Oligonucleotide arrays (HDONAs), such as the Affymetrix HG-U133A GeneChip, use sets of probes chosen to match specified genes, with the expectation that if a particular gene is highly expressed then all the probes in the designated probe set will provide a consistent message signifying the gene's presence. However, we demonstrate by data mining thousands of CEL files from NCBI's GEO database that 4G-probes (defined as probes containing sequences of four or more consecutive guanine (G) bases) do not react in the intended way. Rather, possibly due to the formation of G-quadruplexes, most 4G-probes are correlated, irrespective of the expression of the thousands of genes for which they were separately intended. It follows that 4G-probes should be ignored when calculating gene expression levels. Furthermore, future microarray designs should make no use of 4G-probes.