Reference gene selection for miRNA and mRNA normalization in potato in response to potato virus Y

Mol Cell Probes. 2021 Feb:55:101691. doi: 10.1016/j.mcp.2020.101691. Epub 2020 Dec 25.

Abstract

This was the first report on evaluating candidate reference genes for quantifying the expression profiles of both coding (e.g., mRNA) and non-coding (e.g., miRNA) genes in potato response to potato virus Y (PVY) inoculation. The reverse transcription-quantitative real-time polymerase chain reaction (RT-qPCR) method was employed to quantify the expression profiles of eight selected candidate reference genes; their expression stability was analyzed by four statistical algorithms, i.e., geNorm, BestKeeper, NormFinder and RefFinder. The most stable reference genes were sEF1a, sTUBb and seIF5 with a high stability. The least stable ones were sPP2A, sSUI1 and sGAPDH. The same reference gene allows for normalization of both miRNA and mRNA levels from a single RNA sample using cDNAs synthesized in a single RT reaction, in which a stem-loop primer was used for miRNAs and the oligo (dT) for mRNAs.

Keywords: Hypersensitive resistance; PVY; Protein coding reference gene; Solanum tuberosum; Stem-loop RT-qPCR; microRNA.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Primers / metabolism
  • Gene Expression Profiling
  • Gene Expression Regulation, Plant
  • Genes, Plant*
  • MicroRNAs / genetics*
  • Potyvirus / physiology*
  • RNA, Messenger / genetics*
  • Real-Time Polymerase Chain Reaction / standards*
  • Reference Standards
  • Reproducibility of Results
  • Software
  • Solanum tuberosum / genetics*
  • Solanum tuberosum / virology*

Substances

  • DNA Primers
  • MicroRNAs
  • RNA, Messenger