Spotted long oligonucleotide arrays for human gene expression analysis

Abstract

DNA microarrays produced by deposition (or 'spotting')of a single long oligonucleotide probe for each gene may be an attractive alternative to other types of arrays. We produced spotted oligonucleotide arrays using two large collections of approximately 70-mer probes, and used these arrays to analyze gene expression in two dissimilar human RNA samples. These samples were also analyzed using arrays produced by in situ synthesis of sets of multiple short (25-mer) oligonucleotides for each gene (Affymetrix GeneChips). We compared expression measurements for 7344 genes that were represented in both long oligonucleotide probe collections and the in situ-synthesized 25-mer arrays. We found strong correlations (r = 0.8-0.9) between relative gene expression measurements made with spotted long oligonucleotide probes and in situ-synthesized 25-mer probe sets. Spotted long oligonucleotide arrays were suitable for use with both unamplified cDNA and amplified RNA targets, and are a cost-effective alternative for many functional genomics applications. Most previously reported evaluations of microarray technologies have focused on expression measurements made on a relatively small number of genes. The approach described here involves far more gene expression measurements and provides a useful method for comparing existing and emerging techniques for genome-scale expression analysis.

Figure 1

Differential expression and signal intensity measurements for all three array types. (A) Version 1 spotted long oligonucleotide arrays (means from six replicate two-color hybridizations). (B) Version 2 spotted long oligonucleotide arrays (four replicate two-color hybridizations). (C) In situ-synthesized 25-mer arrays (two replicate K562 sample single-color hybridizations and three replicate pool sample single-color hybridizations). Each point represents data from a single long oligonucleotide probe (A, B) or 25-mer probe set (C). M is a measure of differential gene expression (log₂ [K562 intensity / pool intensity]). A is a measure of signal intensity (0.5 log₂ K562 intensity + 0.5 log₂ pool intensity).

Figure 2

Representation of genes on the three array types. Figures indicate the numbers of genes (distinct UniGene clusters) represented by at least one probe (or probe set) on each of the array types employed in this study. A total of 7344 genes were represented on all three array types.

Figure 3

Comparison of gene expression measurements from Version 1 (v1) long oligonucleotide arrays and in situ-synthesized 25-mer arrays. (A) Differential gene expression measurements for all 7344 common genes. (B) Differential gene expression measurements for 2877 genes remaining after exclusion of probes or probes sets with low signal intensities (A values below the median for that array type). (C) Comparison of signal intensities for all 7344 common genes. (D) Comparison of M values obtained from the first three and the last three v1 oligonucleotide arrays. Each point represents data from a single UniGene cluster. Pearson correlation coefficients (r) are shown for each comparison. Dashed lines are lines of equality.

Figure 4

Comparison of gene expression measurements on Version 2 (v2) long oligonucleotide arrays. Differential expression (A, B) and signal intensity (C, D) measurements from these arrays were compared with measurements from in situ-synthesized 25-mer arrays (A, C) and Version 1 long oligonucleotide arrays (B, D). Comparisons involve the 7344 common genes. For M value comparisons, probes or probe sets with low signal intensity were excluded. After exclusion of those probes, a total of 3133 genes (A) or 3344 genes (B) remained for comparison.

Figure 5

Differential expression measurements for genes with the highest and lowest M values on 25-mer arrays. Genes were divided into subsets according to the M values determined on 25-mer arrays. (A) Genes with M values in the top 1%. (B) Genes with M values close to zero. (C) Genes with M values in the bottom 1%. Each panel shows M values for these subsets of genes as measured using 25-mer arrays (solid lines), Version 1 long oligonucleotide arrays (dotted lines), and Version 2 long oligonucleotide arrays (dashed lines).

Figure 6

Amplified targets used with Version 1 spotted long oligonucleotide arrays. (A) Expression data from six replicate arrays hybridized with cRNA targets produced using a single round of amplification. (B) Data from two replicate arrays with cRNA targets produced using two rounds of amplification. (C, D) Comparisons of M values obtained using unamplified and amplified targets, after exclusion of probes with low A values. After exclusion of low-intensity signals, 5746 probes (C) or 5771 probes (D) of 13,971 total probes remained for comparison. (E, F) Signal intensity values for unamplified and amplified targets. Each point represents data from a single probe.