Methods for detection and quantitation of small RNAs
Improved methods that increase the specificity and sensitivity of detection of small RNAs, including miRNAs, using oligonucleotide primers and nucleic acid amplification, are provided. Reaction conditions that result in preferential decrease in cDNA synthesis of RNAs other than the small RNA molecules targeted for detection during miRNA tailing and reverse transcription reactions are described. Using these reaction conditions greater sensitivity and specificity of amplification of small RNAs including miRNAs is achieved.
1. A method for preparing a cDNA copy of a small RNA molecule, comprising:
(a) providing a small RNA from a biological sample, wherein said RNA is from 18 to 28 nucleotides in length;
(b) incubating the small RNA with an enzyme capable of catalyzing the addition of nucleotides at the 3′ end of the small RNA in the presence of a single ribonucleotide triphosphate selected from the group consisting of ATP, GTP, UTP, and CTP and at a final concentration of divalent magnesium cation between 20 millimolar and 80 millimolar in a reaction to add nucleotides to the small RNA to generate a tailed small RNA;
(c) annealing a DNA primer to the tailed small RNA whereby the DNA template extends from the 3′ end of the tailed small RNA, thereby providing a single stranded region of DNA that may be used to direct polymerization of deoxyribonucleotide triphosphates; and
(d) incubating the annealed tailed small RNA and DNA primer in the presence of reverse transcriptase and deoxyribonucleotide triphosphates and at a final concentration of divalent magnesium cation between 20 millimolar and 80 millimolar under conditions allowing reverse transcription into cDNA and amplification of the annealed tailed small RNA to produce an amplification product.
2. The method of claim 1 , wherein the enzyme used in step (b) is Escherichia coli Poly(A) polymerase.
3. The method of claim 1 , wherein polymerization in step (c) is catalyzed by is MMLV reverse transcriptase.
4. The method of claim 1 , wherein the steps (b), (c) and (d) are performed concurrently in a single reaction mixture.
5. The method of claim 1 , further comprising the step of quantifying the amplification product of step (d).
6. The method of claim 1 , further comprising the step of detecting the amplification product of step (d).
7. The method of claim 4 , further comprising the step of quantifying the amplification product of step (d).
8. The method of claim 4 , further comprising the step of detecting the amplification product of step (d).
9. The method of claim 1 , wherein the final concentration of divalent magnesium cation in steps (b) and (d) is between 30 millimolar and 80 millimolar.
10. The method of claim 1 , wherein the final concentration of divalent magnesium cation in steps (b) and (d) is between 40 millimolar and 80 millimolar.
11. The method of claim 1 , wherein the final concentration of divalent magnesium cation in steps (b) and (d) is between 50 millimolar and 80 millimolar.
12. The method of claim 1 , wherein the final concentration of divalent magnesium cation in steps (b) and (d) is between 70 millimolar and 80 millimolar.