Optimization of gene expression analysis using immobilized capture probes
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.
1. A method of preventing a significant reduction in duplexes detectable in a hybridization assay, the method comprising:
(i) selecting probe lengths for sets of oligonucleotide probes, wherein probes comprise 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 probes attached per unit area on a solid phase carrier which is below a limit at which said significant reduction in detectable duplexes is predicated to take place;
(iii) producing said probes and affixing said probes 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.
2. The method of claim 1 , wherein the density for longer probes is lower than the density for shorter probes.
3. The method of claim 1 , wherein the subsequence of the cognate target is located near the 5′ end of the target.
4. The method of claim 1 further comprising attaching a bifunctional polymeric moiety to the solid phase carriers and then attaching said probes to said bifunctional polymeric moiety.
5. The method of claim 4 , wherein the surface area of the bifunctional polymeric moiety, when attached to the solid phase carriers, is known.
6. The method of claim 4 , wherein said bifunctional polymeric moiety is a polyethylene glycol-having a known approximate molecular weight.
7. The method of claim 4 , wherein said bifunctional polymeric moiety is a protein.
8. The method of claim 7 , wherein the protein is neutravidin.
9. The method of claim 1 , wherein adjacent probe-target complexes attached to the surface do not overlap each other.
10. The method of claim 1 , wherein the probes and the targets are both either RNA or DNA.