Method for Quantification of Small RNA Species
AssigneeExiqon A/S
InventorPeter Kamp Busk
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Abstract
The present invention relates to a method for amplification and quantification of microRNA molecules using quantitative reverse transcription polymerase chain reaction (qRT-PCR) technology. The method comprise the steps of (a) producing cDNA molecules complementary to microRNAs in a sample using polyadenylation and primer extension by reverse transcription, and (b) amplification and quantification of the cDNAs by qPCR using microRNA specific primer sets of forward and reverse primers containing LNA monomers.
Claims51
A method for amplification of a specific RNA molecule in a sample, the method comprising the steps of:
- a) adding poly-A tails to a population of RNA molecules in a sample;
- b) producing cDNA molecules of the poly-A-tailed RNA molecules using an extension primer in a reverse transcription reaction;
- and
- c) amplifying the cDNA molecules by PCR using a forward primer and a reverse primer both of which are specific for said specific RNA molecule,
- wherein said extension primer comprise an anchor sequence.
The method according to claim 46, wherein the extension primer comprises at least one LNA.
The method according to claim 47, wherein the forward primer comprises at least one LNA.
A kit for detecting at least one target RNA comprising at least one primer set specific for the detection of the target RNA, the primer set comprising:
- a) an extension primer of formula III:
- R1-(T)y-R2 (III)
- wherein R1 is a 5′-end nucleotide sequence, (T)y is a central part of y consecutive thymine resides, wherein y is an integer of from 1 to 100, and R2 is a 3′-end nucleotide sequence.
- b) a reverse primer of formula II:
- R3-(T)x-R4 (II)
- wherein R3 is a 5′-end nucleotide sequence, (T)x is a central part of x consecutive thymine residues, wherein x is an integer of from 1 to 100, and R4 is a 3′-end nucleotide sequence that specifically hybridizes to a nucleotide sequence of a target RNA molecule.
- c) a forward primer
A high-throughput method for detecting or measuring the amount of specific target microRNAs in a sample from a living organism, according to claim 51, the method further comprising a distribution step in which the combined poly-A-tailing and reverse transcription reaction is aliquoted into individual wells of a microtiter plate containing microRNA specific primer sets of forward and reverse primers, resulting in the steps of:
- c) adding poly-A tails to a population of small RNA molecules in a sample and producing cDNA molecules of the poly-A-tailed small RNA molecules using an extension primer in a reverse transcription reaction;
- and
- d) pipetting aliquots of the combined poly-A-tailing and reverse transcription reaction into individual wells of a microtiter plate
- e) amplifying specific target microRNAs in individual wells of a microtiter plate containing microRNA specific primer sets
- f) measuring the amount of specific microRNA molecules in individual wells.
- being performed in a fashion compatible with a high-throughput experimental set-up.
An oligonucleotide selected from the group of oligonucleotides disclosed in table 18.
Dependencies are inferred from the claim text.