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Dumbbell-PCR for Discriminative Quantification of a Small RNA Variant

  • Megumi Shigematsu
  • Shozo Honda
  • Yohei KirinoEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1680)

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.

Key words

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

Notes

Acknowledgments

This study was supported by NIH grant (GM106047 to YK).

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Copyright information

© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Computational Medicine Center, Sidney Kimmel Medical CollegeThomas Jefferson UniversityPhiladelphiaUSA
  2. 2.Computational Medicine Center, Sidney Kimmel Medical CollegeThomas Jefferson UniversityPhiladelphiaUSA

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