Identification and selection of optimal reference genes for qPCR-based gene expression analysis in Fucus distichus under various abiotic stresses

PLoS One. 2021 Apr 28;16(4):e0233249. doi: 10.1371/journal.pone.0233249. eCollection 2021.

Abstract

Quantitative gene expression analysis is an important tool in the scientist's belt. The identification of evenly expressed reference genes is necessary for accurate quantitative gene expression analysis, whether by traditional RT-PCR (reverse-transcription polymerase chain reaction) or by qRT-PCR (quantitative real-time PCR; qPCR). In the Stramenopiles (the major line of eukaryotes that includes brown algae) there is a noted lack of known reference genes for such studies, largely due to the absence of available molecular tools. Here we present a set of nine reference genes (Elongation Factor 1 alpha (EF1A), Elongation Factor 2 alpha (EF2A), Elongation Factor 1 beta (EF1B), 14-3-3 Protein, Ubiquitin Conjugating Enzyme (UBCE2), Glyceraldehyde-3-phosphate Dehydrogenase (GAPDH), Actin Related Protein Complex (ARP2/3), Ribosomal Protein (40s; S23), and Actin) for the brown alga Fucus distichus. These reference genes were tested on adult sporophytes across six abiotic stress conditions (desiccation, light and temperature modification, hormone addition, pollutant exposure, nutrient addition, and wounding). Suitability of these genes as reference genes was quantitatively evaluated across conditions using standard methods and the majority of the tested genes were evaluated favorably. However, we show that normalization genes should be chosen on a condition-by-condition basis. We provide a recommendation that at least two reference genes be used per experiment, a list of recommended pairs for the conditions tested here, and a procedure for identifying a suitable set for an experimenter's unique design. With the recent expansion of interest in brown algal biology and accompanied molecular tools development, the variety of experimental conditions tested here makes this study a valuable resource for future work in basic biology and understanding stress responses in the brown algal lineage.

Publication types

  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Fucus* / genetics
  • Fucus* / metabolism
  • Gene Expression Regulation, Plant*
  • Genes, Plant*
  • Plant Proteins* / biosynthesis
  • Plant Proteins* / genetics
  • Real-Time Polymerase Chain Reaction / standards*
  • Reference Standards
  • Stress, Physiological*

Substances

  • Plant Proteins

Grants and funding

The Braybrook group at UCLA is funded by The Department of Cell, Molecular and Developmental Biology and The College of Life Sciences (S.A.B); this work was majorly supported by the U.S. Department of Energy Office of Science, Office of Biological and Environmental Research program under Award Number DE-FC02-02ER63421 and the US Department of Energy (Biological and Environmental Research (BER), the Biological Systems Science Division (BSSD); M.L, S.A.B).