Analyzing messenger RNA and micro RNA in the same reaction mixture
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 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.
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 18 C-27 C.
5. The method according to claim 1 wherein the high temperature is 35 C-60 C.
6. The method according to claim 1 wherein self-complementary stem of the hot start mRNA primer comprises between 6-12 nucleotide base-pairs in length.
7. The method according to claim 1 wherein the self-complementary stem of the hot start mRNA primer is blunt-ended.
8. The method according to claim 1 wherein the self-complementary stem of the hot start mRNA primer comprises a 3′ nucleotide overhang.
9. The method according to claim 5 wherein the high temperature is about 42 C.
10. 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.
11. The method according to claim 10 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.
12. The method according to claim 11 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.
13. The method according to claim 10 wherein the low temperature is 18 C-27 C.
14. The method according to claim 10 wherein the high temperature is 35 C-60 C.
15. The method according to claim 14 wherein the high temperature is about 42 C.
16. The method according to claim 10 wherein self-complementary stem of the hot start mRNA primer comprises between 6-12 nucleotide base-pairs in length.
17. The method according to claim 10 wherein the self-complementary stem of the hot start mRNA primer is blunt-ended.
18. The method according to claim 10 wherein the self-complementary stem of the hot start mRNA primer comprises a 3′ nucleotide overhang.