Methods for detection and quantification of small RNA

1. A method comprising:

(a) contacting a sample to be tested for the presence or absence of a target small RNA with:

(i) a linear first primer comprising a target binding region and a tail region, wherein the target binding region comprises about 12 or fewer nucleotides that are complementary to the small RNA;

(ii) a linear second primer comprising a target binding region and a tail region, wherein the target binding region comprises about 12 or fewer nucleotides that are complementary to the reverse complement of the small RNA,

wherein the tail regions of the first primer, the second primer, or both the first primer and the second primer comprise a first label and a first non-natural base;

(b) extending the first primer under conditions suitable to produce a first reaction product of the small RNA, if present in the sample;

(c) amplifying the first reaction product with the first primer and the second primer under conditions suitable to produce an amplification product, wherein a second non-natural base conjugated to a second label is incorporated into the amplification product opposite the first non-natural base, and wherein the first non-natural base is iso-C or iso-G and the second non-natural base is the other of iso-C or iso-G; and

(d) detecting the amplification products produced in step (c) by observing a signal from the first label, the second label, or both the first label and the second label to determine the presence or absence of the target small RNA in the sample.

2. The method of claim 1 , wherein the target small RNA is from about 16 to about 30 nucleotides in length.

3. The method of claim 1 , wherein the target binding regions of the first primer and the second primer are each from about 7 to about 10 nucleotides in length.

4. The method of claim 1 , wherein the tail regions of the first primer and the second primer are each from about 10 to about 25 nucleotides in length.

5. The method of claim 1 , wherein the tail region of the first primer, the second primer, or both comprises at least two non-natural bases.

6. The method of claim 1 , wherein the tail region of the both primers comprise at least one non-natural base.

7. The method of claim 1 , wherein the first non-natural base is iso-C and the second non-natural base is iso-G.

8. The method of claim 1 , wherein the step of extending is performed at a temperature from about 15° C. to about 50° C.

9. The method of claim 1 , wherein the step of amplifying comprises a first annealing step performed at a temperature from about 35° C. to about 45° C.

10. The method of claim 9 , wherein the step of amplifying comprises subsequent annealing steps performed at a temperature at least about 55° C.

11. The method of claim 1 , wherein the first primer, the second primer, or both are capable of amplifying multiple related small RNA isoforms.

12. The method of claim 1 , wherein the first primer, the second primer, or both are specific for a single small RNA isoform.

13. The method of claim 12 , wherein the single small RNA isoform differs from other isoforms at one or more specific nucleotide positions, and at least the 4 terminal nucleotides at the 3′ end of first primer, the second primer, or both are complementary to the single isoform, but not complementary to the other isoforms.

14. The method of claim 1 , wherein the labels are fluorophores.

15. The method of claim 1 , wherein the first label is a fluorophore and the second label is a quencher.

16. The method of claim 1 , wherein the step of detecting comprises quantifying the amount of the small RNA in the sample.

17. The method of claim 16 , wherein quantifying comprises correlating the amount of signal observed from the first label, the second label or both the first label and the second label, to the amount of small RNA in the sample.

18. The method of claim 1 , wherein the step of detecting comprises determining the melting temperature of the amplification products.

19. A method comprising:

(a) contacting a sample to be tested for the presence or absence of at least two target small RNA isoforms with:

(i) a linear first primer comprising a target binding region and a tail region, wherein the target binding region comprises about 12 or fewer nucleotides that are complementary to at least two small RNA isoforms;

(ii) a linear second primer comprising a target binding region and a tail region, wherein the target binding region comprises about 12 or fewer nucleotides that are complementary to the reverse complement of a first small RNA isoform, and the tail region comprises a first label and a first non-natural base;

(iii) a linear third primer comprising a target binding region and a tail region, wherein the target binding region comprises about 12 or fewer nucleotides that are complementary to the reverse complement of a second small RNA isoform, and the tail region comprises a second label and a second occurrence of the first non-natural base;

(b) extending the first primer under conditions suitable to produce a first reaction product of the at least two small RNA isoforms, if present in the sample;

(c) amplifying the first reaction product, with the first primer, the second primer, and the third primer under conditions suitable to produce amplification products of the first small RNA isoform and the second small RNA isoform, wherein a second non-natural base comprising a third label is incorporated into the amplification products opposite the first non-natural base and wherein the first non-natural base is iso-C or iso-G, and the second non-natural base is the other of iso-C or iso-G; and;

(d) detecting the amplification products produced in step (c) by observing a signal from the first label, the second label, or both the first label and the second label, to determine the presence or absence of the target small RNA isoforms in the sample.