DUMBBELL-PCR: A METHOD TO QUANTIFY SPECIFIC SMALL RNA VARIANTS WITH A SINGLE NUCLEOTIDE RESOLUTION AT TERMINAL SEQUENCES

摘要

A method for specifically and efficiently quantify the expression of targeted RNA variants with specific terminal sequences suitable to identify multiple isoforms bearing complex heterogeneity in terminal sequences by hybridizing a 5'-Dbs-adapter to the 5'-end of target RNAs, wherein the 5'-Dbs-adapter has a stem-loop structure whose protruding 5'-end base-pairs 5'-end of target RNAs, and wherein the loop region of 5'-Dbs-adapter contains a base-lacking spacer which will terminate reverse transcription in a subsequent step; hybridizing a 3'-Db-adapter to the 3'-end of target RNAs, wherein the 3'-Db-adapter has a stem-loop structure whose protruding 3'-end base-pairs 3'-end of target RNAs; ligating the both adapters with target RNAs by Rnl2 ligation to form "dumbbell-like" structure; and, amplifying and quantifying the ligation product by TaqMan RT-PCR.