Small RNA Capture, Detection and Quantification
First Claim
Patent Images
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;
reverse transcribing the polyadenylated mature small RNA using a universal reverse transcription primer, thereby forming a cDNA of the mature small RNA, wherein the universal reverse transcription primer comprises a poly(T) portion and a tail portion, wherein the tail portion comprises a universal primer portion;
ligating a universal ligation adaptor to the 3′
end of the cDNA in the presence of a ligation splint, whereby a cDNA ligation product is formed;
amplifying the cDNA ligation product using a universal forward and reverse primer pair to form an amplification product; and
detecting the amplification product.
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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.
35 Citations
55 Claims
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1. A method for detecting a mature small RNA, the method comprising:
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providing a sample comprising a mature small RNA; polyadenylating the 3′
end of the mature small RNA;reverse transcribing the polyadenylated mature small RNA using a universal reverse transcription primer, thereby forming a cDNA of the mature small RNA, wherein the universal reverse transcription primer comprises a poly(T) portion and a tail portion, wherein the tail portion comprises a universal primer portion; ligating a universal ligation adaptor to the 3′
end of the cDNA in the presence of a ligation splint, whereby a cDNA ligation product is formed;amplifying the cDNA ligation product using a universal forward and reverse primer pair to form an amplification product; and detecting the amplification product. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 25, 26, 27, 28, 29, 30, 31, 32)
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9. A method for detecting a mature small RNA, the method comprising:
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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. - View Dependent Claims (10, 11, 12, 13, 14, 15, 16, 17, 19, 20, 21, 22, 23, 24)
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18. A method for detecting a mature small RNA, the method comprising:
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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, whereby a ligation product is formed;polyadenylating the 3′
end of the mature small RNA;extending the polyadenylated mature small RNA using an oligo(dT) primer, thereby forming an extension product; amplifying the extension product using a universal forward and RT/reverse primer pair to form an amplification product; and detecting the amplification product.
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33. A kit for detecting a mature small RNA, the kit comprising:
- a universal reverse transcription primer, a 3′
universal ligation adaptor, a 3′
ligation splint, a reverse transcriptase, and a ligase, wherein the universal reverse transcription primer comprises a poly(T) portion and a tail portion, wherein the tail portion comprises a universal primer portion. - View Dependent Claims (34, 35, 36, 37, 38, 47, 48, 49, 50, 51)
- a universal reverse transcription primer, a 3′
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39. A kit for detecting a mature small RNA, the kit comprising:
- a universal 5′
ligation adaptor, a 5′
ligation splint, a 3′
ligation adaptor, a 3′
ligation splint, a ligase and instructions for using the kit. - View Dependent Claims (40, 41, 42, 43, 44, 45, 46)
- a universal 5′
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52. A composition comprising:
- a cDNA of a mature small RNA comprising a universal reverse transcription primer sequence at the 5′
end, a 3′
ligation adaptor, a 3′
ligation splint, and a ligase. - View Dependent Claims (54, 55)
- a cDNA of a mature small RNA comprising a universal reverse transcription primer sequence at the 5′
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53. A composition for detecting a mature small RNA, the composition comprising:
- a universal 5′
ligation adaptor, a 5′
ligation splint, a 3′
ligation adaptor, and a 3′
ligation splint.
- a universal 5′
Specification