Development of a versatile, target-oriented tiling microarray assay for measuring allele-specific gene expression

Abstract

In the study of gene expression, it is often desirable to distinguish transcript pools derived from different alleles present in the same organism. We report here an oligonucleotide tiling microarray designed to specifically target 518 single nucleotide polymorphisms (SNPs) between the two sequenced rice (Oryza sativa) subspecies indica and japonica. The tiling array included all 25-mer probes interrogating each SNP by placing the polymorphic site at all 25 possible positions within the probe. Through hybridization to a titration series in which the japonica- and indica-derived cDNA templates were mixed with altering proportions, a regression model was used to screen for diagnostic probe sets for each SNP. Our result indicates that 284 (55%) SNPs have at least one diagnostic probe pair suitable for distinguishing and quantifying the relative abundance of allele-specific transcripts. As a proof-of-concept, we analyzed allele-specific expression in reciprocal indica×japonica F(1) hybrids and detected imbalanced expression at approximately one third of the SNPs. These results were validated by RNA-sequencing and allele-specific real-time PCR experiments. Together, our work demonstrates the utility and advantages of the tiling array method in interrogating large numbers of SNPs for quantifying allele-specific gene expression.