Methods of co-detecting MRNA and small non-coding RNA

摘要

Disclosed herein are methods of co-detecting presence of target messenger RNA (mRNA) and small non-coding RNA (for example, miRNA) in a sample. The disclosed methods can be used to simultaneously detect mRNA and small non-coding RNA in a single assay (for example in the same reaction or the same well of a multi-well assay). The methods can include contacting a sample with a plurality of nuclease protection probes (NPPs) including at least one probe which specifically binds to a target mRNA and at least one probe which specifically binds to a target small non-coding RNA, contacting the sample with a nuclease specific for single-stranded nucleic acids, and detecting the NPP, for example on a microarray.

主权项

1. A method of co-detecting messenger RNA (mRNA) and small non-coding RNA in a sample, comprising:
contacting a sample at about 37-42° C. with at least 10 nuclease protection probes (NPPs), wherein the at least 10 NPPs comprise (i) at least one NPP that specifically binds to at least one target mRNA and comprises a target mRNA-specific portion consisting of 15-30 nucleotides and (ii) at least one NPP that specifically binds to at least one target small non-coding RNA and comprises a target small non-coding RNA-specific portion consisting of 18-23 nucleotides, under conditions sufficient for the NPPs to specifically bind to the target mRNA or target small non-coding RNA, and wherein each of the at least 10 NPPs comprises a detectable label; contacting the sample with a nuclease specific for single-stranded nucleic acid molecules at about 37-50° C. under conditions sufficient to remove unbound nucleic acid molecules; and detecting a presence of at least one NPP that specifically binds to the at least one target mRNA and at least one NPP that specifically binds to the at least one small non-coding RNA, thereby co-detecting the at least one mRNA and the at least one small non-coding RNA in the sample, wherein the at least one mRNA and the at least one small non-coding RNA are detectable in as few as 2000 cells.