Dumbbell-PCR: a method to quantify specific small RNA variants with a single nucleotide resolution at terminal sequences

A method to quantify specific small RNA variants with single nucleotide resolution at a 5′-terminal sequence comprising:

• a) creating the following adapters and primers:
• (i) a 5′-Db-adapter, wherein the nucleotide sequence of the 5′-Db-adapter is SEQ ID NO:
• 2, wherein SEQ ID NO:
• 2 includes a stem-loop structure with the stem formed between nucleotides 7-15 and nucleotides 43-51, and wherein the 5′-terminal six nucleotides of SEQ ID NO:
• 2 are complementary to 5′-terminal sequences of target RNAs;
• (ii) an RT/R primer which is complementary to a 3′-terminal sequence of the target RNAs;
• (iii) a forward primer;
• and (iv) a probe targeting the boundary of a target RNA and the 5′-Db-adapter, wherein the probe contains a 5′ fluorophore and a 3′ quencher;
• b) hybridizing the 5′-Db-adapter to the 5′-end of target RNAs;
• c) ligating the 5′-Db-adapter with target RNAs by Rnl2 ligation to form ligation products;
• and
• d) amplifying and quantifying the ligation products by RT-PCR PCR assay using a DNA polymerase with 5′ exonuclease activity, wherein the probe is designed to target the boundary of the 5′-Db-adapter and target RNAs to exclusively quantify ligated target RNAs.

A method to quantify specific small RNA variants with single nucleotide resolution at a 3′-terminal sequence comprising:

• a) creating the following:
• (i) a 3′-Db-adapter, wherein the nucleotide sequence of the 3′-Db-adapter is SEQ ID NO:
• 1, wherein the SEQ ID NO:
• 1 includes a stem-loop structure with the stem formed between nucleotides 1-8 and 25-32, and wherein the six 3′ nucleotides of SEQ ID NO:
• 1 are complementary to a 3′-terminal sequence of target RNAs;
• (ii) an RT primer;
• (iii) a reverse primer;
• (iv) a forward primer which is complementary to a 5′-terminal sequence of target RNAs;
• and (v) a probe targeting the boundary of the target RNAs and the 3′-Db-adapter, wherein the probe contains a 5′ fluorophore and a 3′ quencher;
• b) hybridizing the 3′-Db-adapter to the 3′-end of the target RNAs;
• c) ligating the 3′-Db-adapter with the target RNAs by Rnl2 ligation to form ligation products;
• and
• d) amplifying and quantifying the ligation products by RT-PCR PCR assay using a DNA polymerase with 5′ exonuclease activity, wherein the probe is designed to target the boundary of the 3′-Db-adapter and target RNAs to exclusively quantify ligated target RNAs.