NOVEL METHODS FOR QUANTIFICATION OF MICRORNAS AND SMALL INTERFERING RNAS
First Claim
1. A method for quantitative determination of a short-length RNA, which has a length of at most 100 nucleotides, comprising the steps of:
- a) preparing, from a sample comprising said short-length RNA, a template polynucleotide which consists of
1) a single stranded target sequence consisting of the sequence of said short-length RNA, its corresponding DNA sequence or a nucleotide sequence complementary to the sequence of said short-length RNA and
2) a 5′ and
/or a 3′
adjacent nucleotide sequence by appending said 5′ and
/or a 3′
adjacent nucleotide sequence to said single stranded target sequence;
b) using said template polynucleotide in a reverse transcription or a nucleotide polymerization to obtain a strand of cDNA; and
c) performing a quantitative real-time PCR (qPCR) including as template(s) said cDNA and optionally the template polynucleotide.
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Abstract
The invention relates to ribonucleic acids, probes and methods for detection, quantification as well as monitoring the expression of mature microRNAs and small interfering RNAs (siRNAs). The invention furthermore relates to methods for monitoring the expression of other non-coding RNAs, mRNA splice variants, as well as detecting and quantifying RNA editing, allelic variants of single transcripts, mutations, deletions, or duplications of particular exons in transcripts, e.g., alterations associated with human disease such as cancer. The invention furthermore relates to methods for detection, quantification as well as monitoring the expression of deoxy nucleic acids.
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Citations
36 Claims
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1. A method for quantitative determination of a short-length RNA, which has a length of at most 100 nucleotides, comprising the steps of:
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a) preparing, from a sample comprising said short-length RNA, a template polynucleotide which consists of
1) a single stranded target sequence consisting of the sequence of said short-length RNA, its corresponding DNA sequence or a nucleotide sequence complementary to the sequence of said short-length RNA and
2) a 5′ and
/or a 3′
adjacent nucleotide sequence by appending said 5′ and
/or a 3′
adjacent nucleotide sequence to said single stranded target sequence;b) using said template polynucleotide in a reverse transcription or a nucleotide polymerization to obtain a strand of cDNA; and c) performing a quantitative real-time PCR (qPCR) including as template(s) said cDNA and optionally the template polynucleotide. - View Dependent Claims (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36)
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Specification