Materials and methods for profiling micrornas
AssigneeUniversity of South Florida
InventorsShyam S. Mohapatra, Jia-Wang Wang
Life of this patent
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Abstract
The present invention provides materials and methods for detecting, quantifying, and/or high-throughput-profiling microRNAs. Advantageously, the present invention is more sensitive and specific than other currently-available miRNA qPCR assays. In addition, the present invention is convenient, easy-to-perform, and cost-effective. In one embodiment, the present invention provides a universal primer for reverse transcription of all miRNAs, a universal reverse primer for PCR amplification reaction, and universal probes. In another embodiment, the present invention provides assays that allow simultaneous detection and/or quantification of a plurality of target miRNAs using a single reverse transcription reaction.
Claims26
A method for detecting, quantifying, and/or profiling a target miRNA, comprising:
- a) contacting a sample containing miRNAs with an effective amount of poly(A)polymerase molecules to yield 3′ end-polyadenylated miRNA molecules,
- b) contacting the sample with an effective amount of a universal primer for reverse transcription and reverse transcriptases, and reverse transcribing the polyadenylated miRNA molecules to yield corresponding c-DNA molecules;
- and
- c) contacting the sample with an effective amount of a universal reverse primer and a target miRNA-specific forward primer, and amplifying the corresponding c-DNA molecules using an amplification reaction;
- wherein the universal primer for reverse transcription is an oligonucleotide comprising:
- a (dT)n sequence flanked by a stem-looped universal adaptor sequence, wherein “n” is an integer ranging from 8 to 50, wherein the universal primer comprises at least two nucleotides adjacent to the 3′ end of the (dT)n sequence, and the nucleotide immediately adjacent to the (dT)n sequence is not T, wherein the universal adaptor sequence near the 5′ end of the (dT)n sequence forms into a stem-loop structure by base-pairing, and
- wherein the universal reverse primer in the amplification reaction is an oligonucleotide comprising a sequence that is, or base-pairs with, at least part of the adaptor sequence near or toward the 5′ end of the (dT)n sequence.
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The method, according to 6, wherein at least two universal probes are used.
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A composition of matter comprising:
- (a) a universal primer for detecting, quantifying and/or profiling miRNA, wherein the universal primer is a primer for reverse transcription of miRNAs, wherein the universal primer is an oligonucleotide comprising:
- a (dT)n sequence flanked by a stem-looped universal adaptor sequence, wherein “n” is an integer ranging from 8 to 50, wherein the universal primer comprises at least two nucleotides adjacent to the 3′ end of the (dT)n sequence, and the nucleotide immediately adjacent to the (dT)n sequence is not T, and wherein the universal adaptor sequence near the 5′ end of the (dT)n sequence forms into a stem-loop structure by base-pairing;
- or
- (b) a universal primer for detecting, quantifying and/or profiling miRNA, wherein the universal primer is a reverse primer for an amplification reaction wherein the universal primer is an oligonucleotide comprising a sequence that is, or base-pairs with, at least part of a stem-looped universal adaptor sequence;
- or
- (c) a universal probe for detecting, quantifying and/or profiling miRNA, wherein the universal probe comprises a sequence that is, or base-pairs with, at least part of a stem-looped universal adaptor sequence;
- or
- (d) a kit for detecting, quantifying and/or profiling a level of expression of miRNA, comprising a universal primer for reverse transcription;
- and a universal reverse primer for an amplification reaction.
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Dependencies are inferred from the claim text.