Optimization of gene expression analysis using immobilized capture probes

Abstract

Disclosed are methods of multiplexed analysis of oligonucleotides in a sample, including a method of preventing a significant reduction in duplexes detectable in a hybridization assay involving (i) selecting probe lengths for sets of oligonucleotide probes, wherein probes include different subsequences such that at least one subsequence is complementary to a subsequence in a cognate target; wherein probes for longer cognate targets are longer in length than probes for shorter cognate targets, (ii) selecting, for each set of probes, a density of oligonucleotides probes attached per unit area on a solid phase carrier which is below a limit at which the significant reduction in detectable duplexes is predicated to take place, (iii) producing the probes and affixing them to different solid phase carriers at the selected density, and (iv) annealing targets to the probes, wherein signal intensities of probes and targets of different lengths are about the same.