Novel methods for quantification of micrornas and small interfering rnas

Abstract

The invention relates to ribonucleic acids, probes and methods for detection, quantification as well as monitoring the expression of mature microRNAs and small interfering RNAs (siRNAs). The invention furthermore relates to methods for monitoring the expression of other non-coding RNAs, mRNA splice variants, as well as detecting and quantifying RNA editing, allelic variants of single transcripts, mutations, deletions, or duplications of particular exons in transcripts, e.g., alterations associated with human disease such as cancer. The invention furthermore relates to methods for detection, quantification as well as monitoring the expression of deoxy nucleic acids.

1 . A method for quantitative determination of a short-length RNA, which has a length of at most 100 nucleotides, comprising the steps of:
 a) contacting the target nucleotide sequence with two oligonucleotide tagging probes each comprising an anchor nucleotide sequence and a recognition nucleotide sequence, wherein said recognition nucleotide sequence is complementary to the target sequence, and wherein the recognition sequence of the first tagging probe hybridizes to a first region of the target sequence and the second recognition sequence of the second tagging probe hybridizes to a second region of the target sequence adjacent to the first region of the target sequence;   b) joining the two adjacent recognition sequences of the hybridized tagging probes covalently by ligation to form a contiguous nucleotide sequence, comprising a sequence complementary to the target nucleotide sequence and the two anchor nucleotide sequences, and   c) quantifying the ligated oligonucleotide molecules by PCR using primers corresponding to the anchor nucleotide sequences and a labelled detection probe comprising a target recognition sequence and a detection moiety.   
     
     
         2 . The method of according to  claim 1 , wherein at least one of the anchor nucleotide sequences is a polynucleotide consisting of identical nucleotides. 
     
     
         3 . The method according to  claim 1 , wherein one or both of the anchor nucleotide sequences do not occur naturally in the organism from where the sample RNA is derived. 
     
     
         4 . The method according to  claim 1 , wherein one of the tagging probes contains a moiety that enables immobilisation onto a solid support. 
     
     
         5 . The method according to  claim 1 , wherein the sample in step (a) is enriched for RNA of short length. 
     
     
         6 . The method according to  claim 1 , wherein the labelled detection probe comprises modified nucleotides. 
     
     
         7 . The method according to  claim 6 , wherein the modified nucleotides are LNA nucleotides. 
     
     
         8 . The method according to  claim 6 , wherein the labelled detection probe corresponds to or is complementary to a sequence in the short-length RNA. 
     
     
         9 . The method according to  claim 1 , wherein primers used in PCR comprise modified nucleotides. 
     
     
         10 . The method according to  claim 9 , wherein the modified nucleotides are LNA nucleotides.