Identification and validation of reference genes for quantitative real-time PCR normalization and its applications in lycium

PLoS One. 2014 May 8;9(5):e97039. doi: 10.1371/journal.pone.0097039. eCollection 2014.

Abstract

Lycium barbarum and L. ruthenicum are extensively used as traditional Chinese medicinal plants. Next generation sequencing technology provides a powerful tool for analyzing transcriptomic profiles of gene expression in non-model species. Such gene expression can then be confirmed with quantitative real-time polymerase chain reaction (qRT-PCR). Therefore, use of systematically identified suitable reference genes is a prerequisite for obtaining reliable gene expression data. Here, we calculated the expression stability of 18 candidate reference genes across samples from different tissues and grown under salt stress using geNorm and NormFinder procedures. The geNorm-determined rank of reference genes was similar to those defined by NormFinder with some differences. Both procedures confirmed that the single most stable reference gene was ACNTIN1 for L. barbarum fruits, H2B1 for L. barbarum roots, and EF1α for L. ruthenicum fruits. PGK3, H2B2, and PGK3 were identified as the best stable reference genes for salt-treated L. ruthenicum leaves, roots, and stems, respectively. H2B1 and GAPDH1+PGK1 for L. ruthenicum and SAMDC2+H2B1 for L. barbarum were the best single and/or combined reference genes across all samples. Finally, expression of salt-responsive gene NAC, fruit ripening candidate gene LrPG, and anthocyanin genes were investigated to confirm the validity of the selected reference genes. Suitable reference genes identified in this study provide a foundation for accurately assessing gene expression and further better understanding of novel gene function to elucidate molecular mechanisms behind particular biological/physiological processes in Lycium.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Fruit / genetics
  • Gene Expression Profiling / standards*
  • Genes, Plant / genetics*
  • Lycium / drug effects
  • Lycium / genetics*
  • Lycium / physiology
  • Organ Specificity
  • Real-Time Polymerase Chain Reaction / standards*
  • Reference Standards
  • Reproducibility of Results
  • Salts / pharmacology
  • Stress, Physiological / drug effects
  • Stress, Physiological / genetics

Substances

  • Salts

Grants and funding

This work was supported by National Natural Science Foundation of China (Grant no.31100223), Scientific Research Equipment Project of Chinese Academy of Sciences (YZ201227), and Key Laboratory of Plant Resources Conservation and Sustainable Utilization. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.