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Small RNA capture, detection and quantification

  • US 9,920,360 B2
  • Filed: 11/04/2013
  • Issued: 03/20/2018
  • Est. Priority Date: 11/02/2012
  • Status: Active Grant
First Claim
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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 reverse primer portion;

    ligating a universal ligation adaptor to the 3′

    end of the cDNA, whereby a cDNA ligation product is formed, wherein the universal ligation adaptor comprises a ligation splint portion and further comprises a stem-loop structure;

    amplifying the cDNA ligation product using a universal forward and reverse primer pair to form an amplification product; and

    detecting the amplification product with a probe specific for the mature small RNA, wherein the ligating and the amplifying occur during overlapping intervals in the same reaction vessel.

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