Quantitative assessment of the use of modified nucleoside triphosphates in expression profiling: differential effects on signal intensities and impacts on expression ratios

Abstract

Abstract Background The power of DNA microarrays derives from their ability to monitor the expression levels of many genes in parallel. One of the limitations of such powerful analytical tools is the inability to detect certain transcripts in the target sample because of artifacts caused by background noise or poor hybridization kinetics. The use of base-modified analogs of nucleoside triphosphates has been shown to increase complementary duplex stability in other applications, and here we attempted to enhance microarray hybridization signal across a wide range of sequences and expression levels by incorporating these nucleotides into labeled cRNA targets. Results RNA samples containing 2-aminoadenosine showed increases in signal intensity for a majority of the sequences. These results were similar, and additive, to those seen with an increase in the hybridization time. In contrast, 5-methyluridine and 5-methylcytidine decreased signal intensities. Hybridization specificity, as assessed by mismatch controls, was dependent on both target sequence and extent of substitution with the modified nucleotide. Concurrent incorporation of modified and unmodified ATP in a 1:1 ratio resulted in significantly greater numbers of above-threshold ratio calls across tissues, while preserving ratio integrity and reproducibility. Conclusions Incorporation of 2-aminoadenosine triphosphate into cRNA targets is a promising method for increasing signal detection in microarrays. Furthermore, this approach can be optimized to minimize impact on yield of amplified material and to increase the number of expression changes that can be detected.