Four-leaf Clover qRT-PCR: A Convenient Method for Selective Quantification of Mature tRNA

RNA Biol. 2015;12(5):501-8. doi: 10.1080/15476286.2015.1031951.

Abstract

Transfer RNAs (tRNAs) play a central role in translation and also recently appear to have a variety of other functions in biological processes beyond translation. Here we report the development of Four-Leaf clover qRT-PCR (FL-PCR), a convenient PCR-based method, which can specifically quantify individual mature tRNA species. In FL-PCR, T4 RNA ligase 2 specifically ligates a stem-loop adapter to mature tRNAs but not to precursor tRNAs or tRNA fragments. Subsequent TaqMan qRT-PCR amplifies only unmodified regions of the tRNA-adapter ligation products; therefore, FL-PCR quantification is not influenced by tRNA post-transcriptional modifications. FL-PCR has broad applicability for the quantification of various tRNAs in different cell types, and thus provides a much-needed simple method for analyzing tRNA abundance and heterogeneity.

Keywords: FL-PCR; RNA; T4 RNA Ligase 2; TaqMan PCR; tRNA; tRNA abundance; tRNA heterogeneity.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cell Line, Tumor
  • Humans
  • Molecular Sequence Data
  • Nucleic Acid Conformation*
  • RNA, Transfer / chemistry*
  • RNA, Transfer / genetics*
  • Reverse Transcriptase Polymerase Chain Reaction / methods*

Substances

  • RNA, Transfer