Analyzing messenger RNA and micro RNA in the same reaction mixture
AssigneeApplied Biosystems, LLC
InventorsKai Qin Lao, Neil A. Straus
Life of this patent
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Abstract
The present teachings provide methods, compositions, and kits for performing primer extension reactions on at least two target polynucleotides in the same reaction mixture. In some embodiments, a reverse transcription reaction is performed on a first target polynucleotide with a hot start primer comprising a self-complementary stem and a loop, and extension products form at high temperatures but extension products form less so at low temperatures since the self-complementary stem of the hot start primer prevents hybridization of the target specific region to the target. However, non-hot start primers with free target specific regions can hybridize to their corresponding targets at the low temperature and extension can happen at the low temperature.
Claims18
A method of forming a messenger RNA (mRNA) primer extension product and a micro RNA primer extension product in the same reaction mixture comprising;
- forming a reverse transcription reaction mixture comprising, a hot start mRNA primer, a micro RNA primer, a target mRNA, a target micro RNA, and a reverse transcribing enzyme, wherein the hot start mRNA primer comprises a self-complementary stem, and a loop, wherein a contiguous portion of the stem and loop forms a target specific region;
- hybridizing the micro RNA primer to the target micro RNA at a low temperature;
- extending the micro RNA primer at the low temperature to form a micro RNA extension product, wherein the self-complementary stem of the hot start mRNA primer is non-denatured;
- raising the temperature to a high temperature, wherein the self-complementary stem of the hot start mRNA primer is denatured;
- hybridizing the target specific region of the hot start mRNA primer to the target mRNA at the high temperature;
- extending the hot start mRNA primer to form a mRNA extension product;
- and, forming the mRNA primer extension product and the micro RNA primer extension product in the same reaction mixture.
A method of quantifying a messenger RNA (mRNA) and a micro RNA in the same reaction mixture comprising:
- forming a reverse transcription reaction mixture comprising a hot start mRNA primer, a micro RNA primer, a target mRNA, a target micro RNA, and a reverse transcribing enzyme, wherein the hot start mRNA primer comprises a self-complementary stem, and a loop, wherein a contiguous portion of the stem and loop forms a target specific region;
- hybridizing the micro RNA primer to the target micro RNA at a low temperature;
- extending the micro RNA primer at the low temperature to form a micro RNA extension product, wherein the self-complementary stem of the hot start mRNA primer is non-denatured;
- raising the temperature to a high temperature, wherein the self-complementary stem of the hot start mRNA primer is denatured;
- hybridizing the target specific region of the hot start mRNA primer to the target mRNA at the high temperature;
- extending the hot start mRNA primer to form a mRNA extension product;
- and, forming the mRNA primer extension product and the micro RNA primer extension product in the same reaction mixture;
- amplifying the mRNA primer extension product and the micro RNA primer extension product in a multiplexed PCR;
- performing a plurality of lower-plex decoding PCRs by performing a first decoding PCR on the mRNA primer extension product and a second decoding PCR on the micro RNA primer extension product;
- and quantifying the mRNA and the micro RNA.
Dependencies are inferred from the claim text.