Pure miRNA Sample Preparation Method
AssigneeLife Technologies Corporation
InventorKai Qin LAO
Life of this patent
Estimating the term…
Abstract
The present teachings provide novel methods, compositions, and kits for analyzing mature micro RNAs (miRNAs). By taking advantage of the observation that most mature miRNAs in cells are tightly associated with RISCs, the present teachings provide approaches for studying mature miRNAs without the complications of additional nucleic acids. For example, in some embodiments the present teachings provide a method of purifying mature miRNAs comprising heating a sample to form a lysate, and, degrading the additional nucleic acids. The resulting mixture lacks the additional nucleic acids, and contains mature miRNAs associated with RISCs. Liberating the mature miRNAs from RISCs, for example by a protease, a detergent, and/or heat, can result in a pure collection of mature miRNAs.
Claims91
A method of purifying mature miRNAs in a sample comprising mature miRNAs and additional nucleic acids, said method comprising;
- lysing the sample;
- degrading the additional nucleic acids;
- and,
- liberating the mature miRNAs.
A method of selectively synthesizing complementary nucleic acids to mature miRNAs in a sample, without synthesizing complementary nucleic acids to additional nucleic acids in the sample, said method comprising;
- lysing the sample to form a collection RISC-protected mature miRNA and a collection of additional nucleic acids;
- degrading the additional nucleic acids, wherein the RISC-protected mature miRNAs are not degraded;
- liberating the miRNAs of the RISC-protected mature miRNAs to form a collection of pure mature miRNAs;
- treating the pure mature miRNAs with at least one primer and dNTPs;
- extending the at least one primer in a primer extension reaction;
- and,
- synthesizing complementary nucleic acids to mature miRNAs in the sample without synthesizing complementary nucleic acids to additional nucleic acids in the sample.
A method of quantitating a plurality of mature miRNAs from a sample comprising;
- lysing the sample to form a plurality of RISC-protected mature miRNA and a plurality of additional nucleic acids;
- degrading the additional nucleic acids, wherein the plurality of RISC-protected mature miRNAs are not degraded;
- removing the miRNAs from the plurality of RISC-protected mature miRNAs to form a plurality of pure mature miRNAs;
- liberating the plurality of pure mature miRNAs with at least one primer, and dNTPs, wherein at least one of the dNTPs comprises a label;
- extending the at least one primer in a primer extension reaction;
- labeling the plurality of pure mature miRNAs from the sample to form a labeled plurality of pure mature miRNAs;
- hybridizing the labeled plurality of pure mature miRNAs to an array;
- and,
- quantitating the plurality of mature miRNA from the sample.
A method of amplifying a plurality of mature miRNAs from a sample comprising;
- lysing the sample to form a plurality of RISC-protected mature miRNA and a plurality of additional nucleic acids;
- degrading the additional nucleic acids, wherein RISC-protected mature miRNAs are not degraded;
- liberating the miRNAs of the RISC-protected mature miRNAs to form a plurality of pure mature miRNAs;
- and,
- amplifying the pure mature miRNAs.
A method of quantitating a plurality of mature miRNAs from a sample comprising;
- lysing the sample to form a plurality of RISC-protected mature miRNAs and a plurality of additional nucleic acids;
- degrading the additional nucleic acids, wherein RISC-protected mature miRNAs are not degraded;
- liberating the miRNAs of the RISC-protected mature miRNAs to form a plurality of pure mature miRNAs;
- and,
- performing a multiplexed reverse transcription reaction on the plurality of pure mature miRNAs, wherein the multiplexed reverse transcription reaction comprises a plurality of stem-loop primers, to form a plurality of extension products;
- dividing the plurality of extension products into a plurality of reaction vessels, wherein a PCR can occur in a distinct reaction vessel, wherein a reaction vessel comprises a primer pair, wherein the primer pair comprises a reverse primer that was encoded by a stem-loop primer corresponding to a mature miRNA species, and a forward primer that comprises a target-specific portion for the mature miRNA species, and a tail portion;
- performing a plurality of PCRs in the plurality of reaction vessels;
- and,
- quantitating each mature miRNA species in each PCR by inclusion of a detector probe.
A composition comprising a collection of RISC-protected mature miRNAs, a collection of additional nucleic acids, and at least one experimentally-added active nuclease.
A composition comprising a collection of RISC-protected mature miRNAs and at least one experimentally-added nuclease that is inactivated.
A kit for purifying miRNAs comprising;
- at least one nuclease;
- and,
- at least one control nucleic acid.
A method of assessing the efficacy of miRNA knock-down with an antagomir comprising;
- treating a sample with an antagomir for a mature target miRNA;
- measuring the amount of mature target miRNA that results from liberation of mature miRNAs from RISCs;
- comparing the amount of mature target miRNA that results from liberation of mature miRNAs from RISCs in the sample with an expectation value;
- and,
- assessing the efficacy of miRNA knock-down with the antagomir.
Dependencies are inferred from the claim text.