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.
Highlights
Brown algae represent one of the five major lineages that have developed a multicellular body organization independently from other species [1,2]
Housekeeping gene sequences previously reported for Ectocarpus siliculosus [13] were aligned against the related species Fucus serratus embryo development transcriptome [36] to identify putative homologs
Primer sets were designed for all nine genes and their efficiencies and melting temperatures determined by Quantitative real-time PCR (qPCR)
Summary
Brown algae represent one of the five major lineages that have developed a multicellular body organization independently from other species (with red algae, plants/green algae, fungi, and metazoans as the other four) [1,2]. Taking into account their divergent evolutionary history [4], importance as an ecological and economical resource [5,6], and their astonishing ecology [7], the brown algae represent a unique and important group to explore. They have been extremely understudied on a molecular level. It is only within the past decades that scientists have begun to explore their rich molecular biology
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