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 ligating 5′ and 3′ ligation adaptors to the 5′ and 3′ ends of the mature small RNAs, respectively, in the presence of 5′ and 3′ semi-degenerate ligation splints to generate a ligation product. Other embodiments comprise reverse transcribing polyadenylated mature small RNA with a universal reverse transcription primer and ligating an adaptor to the 3′ end of the cDNA in the presence of a semi-degenerate ligation splint to generate a cDNA ligation product.

1 . A kit for detecting a mature small RNA, the kit comprising:
 a universal reverse transcription primer, wherein the universal reverse transcription primer comprises a poly(T) portion and a tail portion, wherein the tail portion comprises a universal primer portion;   a reverse transcriptase; and   a ligase.   
     
     
         2 . The kit of  claim 1 , further comprising one or more dNTPs, ATP, a buffer, and a salt of a divalent cation. 
     
     
         3 . The kit of  claim 1 , further comprising a universal 5′ ligation adaptor and a 5′ ligation splint. 
     
     
         4 . The kit of  claim 3 , wherein the 5′ universal ligation adaptor is an RNA oligonucleotide. 
     
     
         5 . The kit of  claim 3 , wherein the 5′ universal ligation adaptor and 5′ ligation splint form a stem-loop structure. 
     
     
         6 . The kit of  claim 5 , wherein the stem-loop structure includes a single-stranded overhang comprising about 3 to about 6 nucleotide bases which hybridize with the 5′ region of the mature small RNA. 
     
     
         7 . The kit of  claim 3 , wherein the 5′ ligation splint is semi-degenerate and comprises a 5′ terminal region comprising 3 to 6 degenerate nucleotide bases that hybridizes with the 5′ end of the mature small RNA and a 3′ region that hybridizes with the 3′ end of the 5′ universal ligation adaptor. 
     
     
         8 . The kit of  claim 3 , wherein the 5′ ligation adaptor includes a universal primer sequence. 
     
     
         9 . The kit of  claim 8 , further comprising a pair of universal primers, wherein a first universal primer is complementary to or includes a portion of the 5′ ligation adaptor, and wherein a second universal primer is complementary to or includes a portion of the universal primer portion of the tail. 
     
     
         10 . The kit of  claim 9 , further comprising a DNApolymerase. 
     
     
         11 . The kit of  claim 1 , further comprising a blocking oligonucleotide. 
     
     
         12 . The kit of  claim 11 , 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. 
     
     
         13 . A kit for detecting a mature small RNA, the kit comprising:
 a universal 5′ ligation adaptor;   a 5′ ligation splint;   a universal 3′ ligation adaptor;   a 3′ ligation splint;   a reverse transcriptase; and   a ligase.   
     
     
         14 . The kit of  claim 13 , wherein the 3′ ligation splint is semi-degenerate and comprises a 3′ terminal region comprising about 3 to about 6 degenerate nucleotide bases and a 5′ region that hybridizes with the 5′ end of the 3′ universal ligation adaptor. 
     
     
         15 . The kit of  claim 13 , wherein the 3′ universal ligation adaptor and the 3′ ligation splint form a stem-loop structure comprising a 3′ single-stranded overhang comprising about 3 to about 6 nucleotide bases for hybridizing to the mature small RNA. 
     
     
         16 . The kit of  claim 13 , further comprising one or more dNTPs, ATP, a buffer, and a salt of a divalent cation. 
     
     
         17 . The kit of  claim 13 , further comprising a pair of universal primers, wherein a first universal primer is complementary to or includes a portion of the universal 5′ ligation adaptor, and wherein a second universal primer is complementary to or includes a portion of the universal 3′ ligation adaptor. 
     
     
         18 . The kit of  claim 17 , further comprising a DNApolymerase. 
     
     
         19 . The kit of  claim 13 , further comprising a blocking oligonucleotide 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. 
     
     
         20 . A method for detecting a mature small RNA, the method comprising:
 providing a sample comprising a mature small RNA;   ligating a universal 5′ ligation adaptor to the 5′ end of the mature small RNA in the presence of a 5′ semi-degenerate ligation splint, and ligating a universal 3′ ligation adaptor to the 3′ end of the mature small RNA in the presence of a 3′ semi-degenerate ligation splint, whereby a ligation product is formed;   reverse transcribing the ligation using a universal RT/reverse primer, whereby an extension product is formed;   amplifying the extension product using a universal forward and RT/reverse primer pair, whereby an amplification product is formed; and   detecting the amplification product.