US 2015/0031573 A1 · MIRNA ANALYSIS METHODS

Abstract

The present invention provides a PCR-free, multiplexed ligation assay for miRNA expression analysis that produces highly quantitative, 10-100 plex miRNA profiling in a single reaction. The inventive methods use a 2-step ligation assay to generate an array of miRNA specific ligation products that can be decoded and quantified by a size discrimination method such as gel electrophoresis or single molecule separation. One embodiment is a low-cost assay that can be performed using standard tools available in nearly all molecular biology laboratories. This assay requires nothing more than a gel apparatus and reader for detection. Other embodiments include use of magnetic beads and other size exclusion apparatus which give increasingly higher sensitivity, lower sample consumption, and reduced processing steps.

Claims22

Dependent claims

A method for detection of one or more target RNA of interest in a sample comprising:

a) obtaining a sample containing one or more target RNA of interest;
b) adding to the sample of a) a sufficient amount of adapter probes and a sufficient amount of a first ligase
c) performing a first ligation step by incubation and allowing the adapter probes to be ligated to the 3′-OH ends of the one or more target RNA of interest in the sample;
d) adding to the sample of b) a sufficient amount of common probes and discrimination probes, and a sufficient amount of a second ligase;
e) performing a second ligation step by incubating the sample of c) for a sufficient amount of time to effect hybridization of probes of d) with ligated target RNA of interest and adapter probes of b) and to allow ligation between the discrimination probes and common probes;
and
f) analysis of the products of by one or more size discrimination methods.

4 more elements

Dependencies are inferred from the claim text.

Mirna analysis methods

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Mirna analysis methods

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Abstract

Claims22