Analyzing messenger RNA and micro RNA in the same reaction mixture
View Patent ↗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.
1. 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 blunt-ended self-complementary stem, and a loop, wherein the blunt-ended self-complementary stem comprises a target specific region and a quencher 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.
2. The method according to claim 1 wherein the micro RNA primer comprises a self-complementary stem, a loop, and a single-stranded target specific region, wherein the self-complementary stem is substantially non-denatured at both the low temperature and the high temperature.
3. The method according to claim 2 wherein the self-complementary stem of the micro RNA primer is 6-12 nucleotide base-pairs in length and the single stranded target specific region is 6-8 nucleotides in length.
4. The method according to claim 1 wherein the low temperature is 18C-27C.
5. The method according to claim 1 wherein the high temperature is 35C-60C.
6. The method according to claim 1 wherein blunt-ended self-complementary stem comprises between 6-12 nucleotide base-pairs in length.
7. A method of forming a first primer extension product and a second primer extension product in the same reaction mixture comprising;
forming a primer extension reaction mixture comprising a first primer, a hot start second primer, a first target, a second target, and a primer extending enzyme, wherein the hot start second primer comprises a blunt-ended self-complementary stem and a loop, wherein the blunt-ended self-complementary stem comprises a target specific region and a quencher region;
hybridizing the first primer to the first target at a low temperature;
extending the first primer at the low temperature to form a first primer extension product, wherein the self-complementary stem of the hot start second primer is non-denatured;
raising the temperature to a high temperature, wherein the self-complementary stem of the hot start second primer is denatured;
hybridizing the target specific region of the hot start second primer to the second target at the high temperature;
extending the hot start second primer at the high temperature to form a second target extension product; and,
forming the first primer extension product and the second primer extension product in the same reaction mixture.
8. The method according to claim 7 wherein the high temperature is 35C-60C.
9. The method according to claim 7 wherein blunt-ended self-complementary stem comprises between 6 and 12 nucleotide base pairs in length.