Dumbbell-PCR for Discriminative Quantification of a Small RNA Variant

Methods Mol Biol. 2018;1680:65-73. doi: 10.1007/978-1-4939-7339-2_4.

Abstract

Cellular RNAs are often expressed as multiple isoforms of complex heterogeneity in both length and terminal sequences. IsomiRs, the isoforms of microRNAs, are such an example. Distinct quantification of each RNA variant is necessary to unravel the biogenesis mechanism and biological significance of heterogenetic RNA expression. Here we describe Dumbbell-PCR (Db-PCR), a TaqMan RT-PCR-based method that distinctively quantifies a specific small RNA variant with single-nucleotide resolution at terminal sequences. Db-PCR enables the quantitative analysis of RNA terminal heterogeneity without performing Next-Generation Sequencing.

Keywords: Db-PCR; Dumbbell-PCR; Small regulatory RNA; T4 RNA Ligase 2; TaqMan qRT-PCR; isomiR; miRNA; tRNA fragment.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Genetic Variation*
  • MicroRNAs / genetics
  • Polymerase Chain Reaction* / methods
  • RNA, Small Untranslated / genetics*
  • Real-Time Polymerase Chain Reaction
  • Sequence Analysis, RNA

Substances

  • MicroRNAs
  • RNA, Small Untranslated