Optimization of oligonucleotide-based DNA microarrays

Abstract

Oligonucleotide-based DNA microarrays are becoming increasingly useful for the analysis of gene expression and single nucleotide polymorphisms. Here we report a systematic study of the sensitivity, specificity and dynamic range of microarray signals and their dependence on the labeling and hybridization conditions as well as on the length, concentration, attachment moiety and purity of the oligonucleotides. Both a controlled set of in vitro synthesized transcripts and RNAs from biological samples were used in these experiments. An algorithm is presented that allows the efficient selection of oligonucleotides able to discriminate a single nucleotide mismatch. Critical parameters for various applications are discussed based on statistical analysis of the results. These data will facilitate the design and standardization of custom-made microarrays applicable to gene expression profiling and sequencing analyses.

Figure 1

Hybridization of in vitro transcribed RNAs to oligonucleotide microarrays. (A) Layout of oligos corresponding to one gene, and fluorescent scan of the hybridization data corresponding to oligos for one of the genes under study (U2AF35). (B) Microarray layout for the five genes under study and fluorescent scan of hybridization data to the complete set of in vitro transcribed RNAs. The layout of oligo lengths and M/MM controls is indicated. The white box surrounds an area where the oligos spotted correspond to regions of the mRNA not present in our in vitro transcripts. (C) As in (B), without RNA corresponding to the gene SXL. (D) As in (B), with only SXL RNA.

Figure 2

Variation of specificity and sensitivity of the microarray with hybridization temperature and formamide concentration. (A) Variation of M/MM ratios with temperature. Average M/MM ratios for 30-nt oligos corresponding to SXL at the indicated temperatures. Standard deviations are represented by vertical bars. Filled squares represent values at 50% formamide; triangles represent the value at 58% formamide. (B) Variation of signal intensities with temperature. Average fluorescent intensities for 30-nt oligos corresponding to SXL at the indicated temperatures. Standard deviations are represented by vertical bars. Filled squares represent values at 50% formamide; triangles represent the value at 58% formamide.

Figure 3

Hybridization of fluorescently labeled HeLa mRNAs to oligonucleotide microarrays. Microarray layout for the five genes under study, and fluorescent scan of hybridization data to labeled HeLa mRNAs, obtained under optimized conditions.