Patent ID: 8206904

Claim:
A method for detecting an miRNA target, comprising: a) providing: i) a sample suspected of containing an miRNA target, said miRNA target comprising a first region, a second region, and a third region, wherein said first region is downstream of and contiguous to said second region, and wherein said second region is downstream of and contiguous to said third region; ii) a first oligonucleotide that comprises a 3′ portion that is complementary to said first region of said miRNA target, and a 5′ portion that is not complementary to said miRNA target; iii) a second oligonucleotide that comprises a 3′ portion that is substantially homologous to a said third region of said miRNA target and a 5′ portion that is not homologous to said third region of said miRNA target; iv) a reverse transcriptase; v) a DNA polymerase; vi) a 5′ nuclease; vii) a probe oligonucleotide, wherein at least a portion of said probe oligonucleotide is complementary to at least a portion of said second region of said miRNA target; and viii) a first stacker oligonucleotide comprising a 5′ terminal portion, wherein at least the 5′ terminal portion of said stacker oligonucleotide is complementary to a region in said 5′ portion of said first oligonucleotide adjacent to said 3′ portion of said first oligonucleotide; b) incubating components (i) through (viii) under conditions such that an invasive cleavage structure forms and is cleaved, wherein under said conditions: 1) said first oligonucleotide hybridizes to said miRNA target and said first stacker oligonucleotide and is extended by said reverse transcriptase to produce a first extended product complementary to said miRNA target; 2) said first extended product is amplified by polymerase chain reaction using said first oligonucleotide and said second oligonucleotide as primers to form an miRNA cDNA target; 3) said probe oligonucleotide and said first oligonucleotide hybridize to said miRNA cDNA target to form an invasive cleavage structure; and 4) said probe oligonucleotide in said invasive cleavage structure is cleaved by said 5′ nuclease to produce non-target cleavage product; and c) detecting cleavage of said probe oligonucleotide, wherein cleavage of said probe oligonucleotide is indicative of the presence of said miRNA target.