Optimization of gene expression analysis using immobilized capture probes
View Patent ↗Disclosed are methods of multiplexed analysis of oligonucleotides in a sample, including: methods of probe and target “engineering”, as well as methods of assay signal analysis relating to the modulation of the probe-target affinity constant, K by a variety of factors including the elastic properties of target strands and layers of immobilized (“grafted”) probes; and assay methodologies relating to: the tuning of assay signal intensities including dynamic range compression and on-chip signal amplification; the combination of hybridization-mediated and elongation-mediated detection for the quantitative determination of abundance of messages displaying a high degree of sequence similarity, including, for example, the simultaneous determination of the relative expression levels, and identification of the specific class of, untranslated AU-rich subsequences located near the 3′ terminus of mRNA; and a new method of subtractive differential gene expression analysis which requires only a single color label.
1. A method comprising:
a) Providing a sample;
b) Providing probe reagents complementary to a target polynucleotide in the sample;
c) Estimating the concentration of the target in the sample;
d) Adjusting the affinity constant (K) governing the interaction of the target with the probe reagents by:
i. using short target length when the target is anticipated to be present in low concentration; or
ii. using long target length when the target is anticipated to be present in high concentration; wherein the relationship between target length (I) and K is: K=al x : wherein a is a constant and x<0; and
e) Quantifying the target using one or more probe reagents.
2. The method of claim 1 wherein the target polynucleotide is a cDNA produced from a corresponding mRNA by reverse transcription and target length is adjusted by adjusting the placement of a reverse transcription primer on the mRNA.
3. The method of claim of 2 , wherein said primer is placed towards the 5′ terminus of the mRNA.
4. The method of claim 2 , wherein said primer is placed adjacent to the 5 ′ terminus of the mRNA.
5. The method of claim 1 , wherein quantification occurs directly or following an in-vitro nucleic acid amplification reaction.
6. The method of claim 1 , wherein said short target length is less than about 150 nucleotides.
7. The method of claim 1 , wherein said long target length is more than about 150 nucleotides.
8. The method of claim 1 , wherein said probe reagents comprise microparticles.
9. The method of claim 8 , wherein said microparticles are encoded.