RT-qPCR normalization genes in the red alga Chondrus crispus

PLoS One. 2014 Feb 3;9(2):e86574. doi: 10.1371/journal.pone.0086574. eCollection 2014.

Abstract

Chondrus crispus is a common red macroalga living on the rocky shores of the North Atlantic Ocean. It has a long research history, being a major source of carrageenan, a thickener widely used in the food industry, but also for physiological and ecological studies. To establish it as a model for red algae, its genome has been sequenced, allowing the development of molecular tools such as quantification of gene expression, including RNAseq and RT-qPCR. To determine appropriate genes for RT-qPCR normalization, the expression of 14 genes was monitored in 18 conditions using two sets of algal samples: samples from the sequenced strain, cultured and stressed in laboratory conditions and C. crispus collected on the shore and stressed in situ. The expression stability of the genes between the samples was evaluated by comparing the Ct range and using the programs geNorm and NormFinder. The candidate genes encoded translation related proteins (initiation factors IF4A-1 and IF4A-2, elongation factor EF1α and eRF3, an eukaryotic polypeptide chain release factor), cytoskeleton proteins (two β-tubulins, α-tubulin and actin), enzymes involved in the pentose phosphate pathway (glucose 6-phosphate deshydrogenase), protein recycling process (ubiquitin and ubiquitin-conjugating enzyme) and glycolysis (isocitrate dehydrogenase). The two sets of samples showed different expression patterns. Most of the genes were stable in the algae cultivated in the laboratory, whereas environmental samples showed a more important variation in gene expression. When analyzing the two sets separately, the ranking of the most stables genes were different from one method to another. When considering all samples, the two statistical methods were concordant, revealing translation initiation factor 4A-2 and eukaryotic polypeptide chain release factor 3 as pertinent normalization genes. This study highlights thus the importance of testing reference genes according to the experiments as well as the genetic and physiological background of the organism.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Algal Proteins / genetics*
  • Chondrus / genetics*
  • Gene Expression Profiling / methods
  • Gene Expression Profiling / standards*
  • Gene Expression Regulation, Plant*
  • RNA, Plant / genetics
  • RNA, Plant / metabolism
  • Real-Time Polymerase Chain Reaction / methods
  • Real-Time Polymerase Chain Reaction / standards*
  • Reference Values
  • Reproducibility of Results

Substances

  • Algal Proteins
  • RNA, Plant

Grants and funding

This work was supported by funding from IDEALG Grants ANR-10-BTBR-04-02 and 04-04 “investissements d'avenir,” Biotechnologies-Bioressources. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.