Patent ID: 9150919
Filing Date: 2015-10-06
Classification: C12Q

Abstract:
1. A method for preferentially amplifying 3′ mature miRNA (miR-3p) or 5′ mature miRNA (miR-5p) in a sample comprising precursor miRNA strands and single strands of mature miR-3p, mature miR-5p or both, wherein the precursor miRNA is an intermediate cleaved product in a microRNA maturation pathway in which (a) primary miRNA with a hairpin secondary structure comprising a double stranded stem embedding a 3′ mature miRNA sequence and a 5′ mature miRNA sequence and having a 5′ and a 3′ single strand overhang at one end of the stem and a single strand loop at a second end of the stem is first cleaved to produce (b) an intermediate precursor miRNA product with a shortened double stranded stem comprising a 5′ mature miRNA-5p sequence and a 3′ mature miRNA-3p sequence, and the intermediate precursor miRNA product is then cleaved at the loop end to produce (c) 3′ mature miRNA (miR-3p) and 5′ mature miRNA (miR-5p) with the mature miR-3p having a nucleotide sequence that is the same as a nucleotide sequence in a 3′ stem end part of the intermediate precursor miRNA and with the mature miR-5p having a nucleotide sequence that is the same as a nucleotide sequence in a 5′ stem end part of the precursor miRNA; the method comprising the steps of: (i) providing the sample comprising precursor miRNA strands and single strands of mature miR-3p, mature miR-5p or both; (ii) a first reaction step comprising (a) reverse transcribing either the miR-3p strands or the miR-5p strands to form cmiDNA strands that are complementary to miR-3p strands or miR-5p strands comprising a target sequence and (b) nicking, displacing and amplifying single strand copies of the cmiDNA strands such that cmiDNA thus formed that are transcribed from mature miR-3p strands or mature miR-5p strands in which the target sequence has a free 3′-OH not flanked by additional sequences at the 3′ end are displaced and amplified, while cmiDNA strands that are formed from miRNA sequence embedded in the precursor miRNA strands that do not have a free 3′-OH flanked by additional sequences at their 3′ end are not displaced and amplified; and (iii) a second reaction step comprising (a) forming from the cmiDNA strands displaced and amplified in step (ii) miDNA strands that are complementary to the cmiDNA strands and (b) nicking, displacing and amplifying miDNA strands that are formed from cmiDNA strands having a free 3′-OH not flanked by additional sequences at their 3′ end, without nicking, displacing and amplifying miDNA strands that are formed from cmiDNA strands that do not have a free 3′-OH flanked by additional sequences at their 3′ end whereby the miDNA strands so nicked, displaced and amplified comprise a DNA sequence that is a counterpart to the RNA sequence derived from the mature microRNA miR-3p strands or mature miR-5p strands and is not a counterpart to RNA sequences derived from mature miRNA sequences embedded in the intermediate precursor miRNA.