Evaluation of Affymetrix HuSNP chip for analysis of allelic gene expression. (A) Scatter plots for duplicate experiments. In this example, we performed duplicate experiments by using the same genomic DNA (left) or cDNA made from kidney RNA (right). Each circle represents a pair of intensity values in the two experiments for one SNP. The mean fluorescent intensities of the perfect match probe minus the mismatch probe from experiment 2 are plotted against the mean fluorescent intensity of the perfect match probe minus the mismatch probe from experiment 1. The Pearson correlation coefficients of the duplicate experiments for genomic DNAs and cDNA are 0.98 and 0.95, respectively. The P values for both correlation coefficients are <0.0001. In other duplicate sample tests, Pearson correlation coefficients ranged from 0.98–0.99 for genomic DNA and from 0.88 to 0.96 for cDNA, and the P values for these correlation coefficients are also <0.0001. (B) Probe images of the PEG3 gene (SNP rs3143). The probe images were generated by using the Affymetrix MAS 4.0 software. The genomic DNA, kidney cDNA, and liver cDNA from the same fetus are each represented by a set of 16 hybridization signals. Within each set, each individual grid corresponds to a probe. The eight probes for allele A are on the left, and the eight probes for allele B are on the right. The top eight probes are for perfect match probes, whereas the bottom eight probes are for mismatch probes. The genomic DNA hybridized strongly to the perfect match probes for both alleles, whereas cDNAs hybridized strongly to the perfect match probes of allele B only. (C) SNPs/genes on the HuSNP chip are summarized in this diagram. There are 1494 SNPs on the HuSNP chip. We mapped 1063 SNPs to transcribed regions and 431 SNPs outside of the transcribed regions. Of the 1063 SNPs, 602 SNPs were analyzed (for selection criteria, see Methods). Among these 602 SNPs, 277 of them showed almost equal expression levels between the two alleles, whereas 156 SNPs had a ratio of gene expression between twofold and fourfold, and 170 SNPs had a ratio exceeding fourfold for at least one individual.
