title: Dumbbell-PCR: a method to quantify specific small RNA variants with a single nucleotide resolution at terminal sequences.
abstract: Recent advances in next-generation sequencing technologies have revealed that cellular functional RNAs are not always expressed as single entities with fixed terminal sequences but as multiple isoforms bearing complex heterogeneity in both length and terminal sequences, such as isomiRs, the isoforms of microRNAs. Unraveling the biogenesis and biological significance of heterogenetic RNA expression requires distinctive analysis of each RNA variant. Here, we report the development of dumbbell PCR (Db-PCR), an efficient and convenient method to distinctively quantify a specific individual small RNA variant. In Db-PCR, 5'- and 3'-stem-loop adapters are specifically hybridized and ligated to the 5'- and 3'-ends of target RNAs, respectively, by T4 RNA ligase 2 (Rnl2). The resultant ligation products with 'dumbbell-like' structures are subsequently quantified by TaqMan RT-PCR. We confirmed that high specificity of Rnl2 ligation and TaqMan RT-PCR toward target RNAs assured both 5'- and 3'-terminal sequences of target RNAs with single nucleotide resolution so that Db-PCR specifically detected target RNAs but not their corresponding terminal variants. Db-PCR had broad applicability for the quantification of various small RNAs in different cell types, and the results were consistent with those from other quantification method. Therefore, Db-PCR provides a much-needed simple method for analyzing RNA terminal heterogeneity.
authors.affiliation: Computational Medicine Center, Sidney Kimmel Medical College, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA.
authors.forename: Shozo
authors.initials: S
authors.lastname: Honda
authors.affiliation: Computational Medicine Center, Sidney Kimmel Medical College, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA Yohei.Kirino@jefferson.edu.
authors.forename: Yohei
authors.initials: Y
authors.lastname: Kirino
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D045744:Cell Line, Tumor; D014644:Genetic Variation; D006801:Humans; D035683:MicroRNAs / Q000032:analysis / Q000737:chemistry; D009690:Nucleic Acid Conformation; D058727:RNA, Small Untranslated / Q000032:analysis* / Q000737:chemistry*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: C505362:MIRN16 microRNA, human; D035683:MicroRNAs; D058727:RNA, Small Untranslated