Dumbbell-PCR: a method to quantify specific small RNA variants with a single nucleotide resolution at terminal sequences.
Bottom Line: 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).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.
Affiliation: Computational Medicine Center, Sidney Kimmel Medical College, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA.Show MeSH
Mentions: We designed methods of 3′-Db-PCR (Figure 1A) and 5′-Db-PCR (Figure 1B) for selective quantification of specific 3′- and 5′-variant of small RNAs, respectively. In 3′-Db-PCR, total RNA is extracted from cells and a 3′-Db-adapter containing protruding 3′-end is hybridized to the 3′-end of the target RNA in the total RNA. In 5′-Db-PCR, a 5′-Db-adapter containing protruding 5′-end is hybridized to the 5′-end of the target RNA. For subsequent Rnl2 ligation, the target RNA in 3′- or 5′-Db-PCR should contain a 3′-OH or 5′-P end, respectively. If the target RNA is expected to contain a different terminal structure, total RNA should be subjected to dephosphorylation/phosphorylation treatment in advance. If the hybridized RNA is the target RNA with exact terminal sequences, the hybridization unites the Db-adapters and the target RNA to form double-stranded nucleotides containing a nick, which is an efficient substrate for Rnl2 ligation (40–42). Following hybridization, Rnl2 ligates the adapter to the target RNA to generate a ligation product with a one-sided ‘dumbbell-like’ secondary structure. Rnl2 ligation efficiency becomes severely reduced when double-stranded nucleotides of the substrate contain gaps or overlaps (41), suggesting that Rnl2 ligation achieves high specificity toward target RNA. Finally, the ligation product is amplified and quantified by TaqMan RT-PCR. The TaqMan probe is designed to target the boundary of the adapter and target RNA for the PCR to exclusively quantify ‘dumbbell-like’ ligation products. Because the TaqMan probe has the ability to discriminate difference of a single nucleotide (47), the design results in highly specific detection of target RNA, which does not cross-react with its 3′- or 5′-terminal variants. Hence, 3′- or 5′-Db-PCR results in highly specific detection and quantification of specific 3′- or 5′-variant of RNAs with single-nucleotide discrimination ability.
Affiliation: Computational Medicine Center, Sidney Kimmel Medical College, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA.