Methods for quantitating small rna molecules

Abstract

In one aspect, the present invention provides methods for amplifying a microRNA molecule to produce DNA molecules. The methods each include the steps of: (a) using primer extension to make a DNA molecule that is complementary to a target microRNA molecule; and (b) using a universal forward primer and a reverse primer to amplify the DNA molecule to produce amplified DNA molecules. In some embodiments of the method, at least one of the forward primer and the reverse primer comprise at least one locked nucleic acid molecule.

1 . A kit for detecting at least one human microRNA selected from the group consisting of miR-21, miR-22, miR-33, miR-34a, miR-34b, miR-34c, miR-122, and miR-122a, the kit comprising at least one or more oligonucleotide primers selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:8; SEQ ID NO:20, SEQ ID NO:147 and SEQ ID NO:148. 
     
     
         2 . The kit according to  claim 1 , comprising at least one primer selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:73 and SEQ ID NO:74 for detecting miR-21. 
     
     
         3 . The kit according to  claim 1 , comprising at least one primer selected from the group consisting of SEQ ID NO:449 and SEQ ID NO:450 for detecting miR-22. 
     
     
         4 . The kit according to  claim 1 , comprising at least one primer selected from the group consisting of SEQ ID NO:111 and SEQ ID NO:112 for detecting miR-33. 
     
     
         5 . The kit according to  claim 1 , comprising at least one primer selected from the group consisting of SEQ ID NO: 113,and SEQ ID NO:114 for detecting miR-34a. 
     
     
         6 . The kit according to  claim 1 , comprising at least one primer selected from the group consisting of SEQ ID NO:115 and SEQ ID NO:116 for detecting miR-34b. 
     
     
         7 . The kit according to  claim 1 , comprising at least one primer selected from the group consisting of SEQ ID NO:117 and SEQ ID NO:118 for detecting miR-34c. 
     
     
         8 . The kit according to  claim 1 , comprising at least one primer selected from the group consisting of SEQ ID NO:8 and SEQ ID NO:20 for detecting miR-122. 
     
     
         9 . The kit according to  claim 1 , comprising at least one primer selected from the group consisting of SEQ ID NO:147 and 148 for detecting miR-122a. 
     
     
         10 . An oligonucleotide primer for detecting a human microRNA selected from the group consisting of SEQ ID NO:6, SEQ ID NO:18, SEQ ID NO:73, SEQ ID NO:74, SEQ ID NO:449, SEQ ID NO:450, SEQ ID NO:111, SEQ ID NO:112, SEQ ID NO: 113, SEQ ID NO:114, SEQ ID NO:115, SEQ ID NO:116, SEQ ID NO:117, SEQ ID NO:118, SEQ ID NO:8; SEQ ID NO:20, SEQ ID NO:147 and SEQ ID NO:148.