Methods, compositions and kits for small rna capture, detection and quantification

Abstract

Methods, compositions and kits for capturing, detecting and quantifying mature small RNAs are provided herein. Embodiments of the methods comprise tailing both the 5′ and 3′ ends of mature small RNA by ligating a 5′ ligation adaptor to the 5′ end and polyadenylating the 3′ end. Other embodiments comprise reverse transcribing the adaptor ligated, polyadenylated mature small RNA with a universal reverse transcription primer and amplifying the cDNA with universal primers.

1 . A method for detecting a mature small RNA, the method comprising:
 providing a sample comprising a mature small RNA;   polyadenylating the 3′ end of the mature small RNA and ligating a single-stranded adaptor to the 5′ end of the mature small RNA in the presence of single strand RNA ligase, whereby an RNA ligation product is formed, wherein the adaptor comprises a universal forward primer portion;   reverse transcribing the RNA ligation product using a reverse transcription (RT) primer, thereby forming a cDNA product of the RNA ligation product, wherein the RT primer comprises a poly(T) portion and a tail portion, wherein the tail portion comprises a universal reverse primer portion;   amplifying the cDNA product using a first forward and reverse primer pair to form an amplification product, wherein the first forward primer can hybridize to the universal forward primer portion or its complement, and the first reverse primer can hybridize to the universal reverse primer portion or its complement; and   detecting the amplification product corresponding to the mature small RNA via quantitative real-time polymerase chain reaction (qPCR).   
     
     
         2 - 32 . (canceled) 
     
     
         33 . A kit for synthesizing and amplifying a mature small RNA cDNA, the kit comprising:
 a single-stranded adaptor comprising a 3′ terminal —OH group and a universal forward primer portion;   a reverse transcription (RT) primer, wherein the RT primer comprises a poly(T) portion and a tail portion and wherein the tail portion comprises a universal reverse primer portion;   a universal forward and reverse primer pair, wherein the universal forward primer can hybridize to the universal forward primer portion or its complement, and the universal reverse primer can hybridize to the universal reverse primer portion or its complement.   
     
     
         34 . The kit of  claim 33 , further comprising one or more dNTPs, ATP, a buffer, and a salt of a divalent cation. 
     
     
         35 . A composition for detecting a mature small RNA comprising:
 a cDNA of a mature small RNA, the cDNA comprising a reverse transcription (RT) primer sequence at the 5′ end and an adaptor sequence at the 3′ end, wherein the RT primer comprises a poly(T) portion and a tail portion and wherein the tail portion comprises a universal reverse primer portion, and wherein the adaptor comprises a universal forward primer portion.   
     
     
         36 . The composition of  claim 35 , further comprising a universal forward and reverse primer pair, wherein the universal forward primer can hybridize to the universal forward primer portion or its complement, and the universal reverse primer can hybridize to the universal reverse primer portion or its complement. 
     
     
         37 . The composition of  claim 36 , further comprising a DNA polymerase. 
     
     
         38 . The kit of  claim 33 , wherein the single-stranded adaptor comprising a 3′ terminal —OH group and a universal forward primer portion is provided in solution. 
     
     
         39 . The kit of  claim 33 , wherein the adaptor is an RNA molecule. 
     
     
         40 . The kit of  claim 33 , further comprising one or more blocking oligonucleotides. 
     
     
         41 . The kit of  claim 40 , wherein the blocking oligonucleotide is selected from the group consisting of a 3′-MGB blocking oligonucleotide, a 5′-MGB blocking oligonucleotide, a 2′-O-methyl blocking oligonucleotide, a 3′-acridine blocking oligonucleotide, a 5′-acridine blocking oligonucleotide, a STAR blocking oligonucleotide, and a blocking oligonucleotide comprising a poly(A) sequence. 
     
     
         42 . The kit of  claim 40 , further comprising a single strand RNA ligase. 
     
     
         43 . The kit of  claim 40 , further comprising a reverse transcriptase. 
     
     
         44 . The kit of  claim 40 , further comprising a DNA polymerase. 
     
     
         45 . The kit of  claim 40 , wherein the poly(T) portion is disposed 3′ of the tail portion in the RT primer. 
     
     
         46 . The kit of  claim 45 , wherein the includes one additional nucleotide 3′ of the poly(T) portion that is not a T. 
     
     
         47 . The kit of  claim 40 , wherein the RT primer is between 15 and 25 nucleotides in length. 
     
     
         48 . The kit of  claim 40 , further comprising one or more additional primer pairs specific to a portion of the cDNA transcribed from a mature small RNA. 
     
     
         49 . The kit of  claim 40 , further comprising a labelled detector probe. 
     
     
         50 . The kit of  claim 40 , further comprising a Poly(A) polymerase. 
     
     
         51 . The kit of  claim 40 , wherein the RNA ligase is RNA ligase I.