Normalisation genes for expression analyses in the brown alga model Ectocarpus siliculosus

Abstract

BackgroundBrown algae are plant multi-cellular organisms occupying most of the world coasts and are essential actors in the constitution of ecological niches at the shoreline. Ectocarpus siliculosus is an emerging model for brown algal research. Its genome has been sequenced, and several tools are being developed to perform analyses at different levels of cell organization, including transcriptomic expression analyses. Several topics, including physiological responses to osmotic stress and to exposure to contaminants and solvents are being studied in order to better understand the adaptive capacity of brown algae to pollution and environmental changes. A series of genes that can be used to normalise expression analyses is required for these studies.ResultsWe monitored the expression of 13 genes under 21 different culture conditions. These included genes encoding proteins and factors involved in protein translation (ribosomal protein 26S, EF1alpha, IF2A, IF4E) and protein degradation (ubiquitin, ubiquitin conjugating enzyme) or folding (cyclophilin), and proteins involved in both the structure of the cytoskeleton (tubulin alpha, actin, actin-related proteins) and its trafficking function (dynein), as well as a protein implicated in carbon metabolism (glucose 6-phosphate dehydrogenase). The stability of their expression level was assessed using the Ct range, and by applying both the geNorm and the Normfinder principles of calculation.ConclusionComparisons of the data obtained with the three methods of calculation indicated that EF1alpha (EF1a) was the best reference gene for normalisation. The normalisation factor should be calculated with at least two genes, alpha tubulin, ubiquitin-conjugating enzyme or actin-related proteins being good partners of EF1a. Our results exclude actin as a good normalisation gene, and, in this, are in agreement with previous studies in other organisms.

Auxin Metabolism and Function in the Multicellular Brown AlgaEctocarpus siliculosus

“…In addition to DNase-I treatment, remnants of genomic DNA contaminant were quantified by amplification of an intron and subtracted from the other values. EsEF1a was chosen as a constitutively expressed gene based on the study by Le Bail et al (2008b) and used for transcript-level normalization. The normalized data were expressed as means 6 SD calculated from the three independent biological experiments.…”

“…The levels of transcripts of EsGRP1 were quantified by real-time reverse transcription-PCR on three independent biological replicates. Each transcript level was normalized to EsEF1a transcripts, as recommended by Le Bail et al (2008b), and averaged (SD indicated). Asterisks indicate P , 0.05 with a t test.…”

Prostaglandin A2triggers a strong oxidative burst in Laminaria: a novel defense inducer in brown algae?

“…The specificity, efficiency and dynamic range of the primers were tested on 1/5 serial dilutions of cDNA (range, 0.24-150 ng for elongation factor 1α [EF1a], vanadium-dependent bromoperoxidase [vBPO], manganese superoxide dismutase [MnSOD] and heat shock protein 70 [HSP70]; 0.12-75 ng for glutaredoxin [GRX]; 0.048-30 ng for glutathione S-transferase [GST]). The absence of genomic DNA contamination was examined in each sample with the intron-specific primer mN (Table 1) (Le Bail et al 2008).…”

“…It has some significant advantages as an experimental model and constitutes one of the beststudied seaweeds (Peters et al 2004, Charrier et al 2008; it can easily be cultivated in small volumes of seawater media both axenically and with associated bacteria (Mül-ler et al 2008); it belongs to a sister group of the ecologically and economically very important kelps; its entire, well-known life cycle can be completed within a few months in culture; many molecular tools are available, including mutant collections (Peters et al 2004), microarrays (Dittami et al 2009) and proteomics . It has also recently become the first seaweed of which the entire genome has been sequenced and thus offers unprecedented opportunities for study (Cock et al 2010).…”

“…The specificity, efficiency and dynamic range of the primers were tested on 1/5 serial dilutions of cDNA (range, 0.24-150 ng for elongation factor 1α [EF1a], vanadium-dependent bromoperoxidase [vBPO], manganese superoxide dismutase [MnSOD] and heat shock protein 70 [HSP70]; 0.12-75 ng for glutaredoxin [GRX]; 0.048-30 ng for glutathione S-transferase [GST]). The absence of genomic DNA contamination was examined in each sample with the intron-specific primer mN (Table 1) (Le Bail et al 2008).Quantitative expression analysis was performed using a QUANTICA™ real-time PCR system (Techne-Barloworld, Stone, Staffordshire, UK). Quantitative polymerase chain reactions (qPCRs) were run in a final volume of 20 µL with 2 ng cDNA and 300 μM of each primer with MesaGreen™ reaction mix (Eurogentec, Southampton, UK).…”