Screening for Suitable Reference Genes for Quantitative Real-Time PCR in Heterosigma akashiwo (Raphidophyceae)

Abstract

The raphidophyte Heterosigma akashiwo is a globally distributed harmful alga that has been associated with fish kills in coastal waters. To understand the mechanisms of H. akashiwo bloom formation, gene expression analysis is often required. To accurately characterize the expression levels of a gene of interest, proper reference genes are essential. In this study, we assessed ten of the previously reported algal candidate genes (rpL17-2, rpL23, cox2, cal, tua, tub, ef1, 18S, gapdh, and mdh) for their suitability as reference genes in this species. We used qRT-PCR to quantify the expression levels of these genes in H. akashiwo grown under different temperatures, light intensities, nutrient concentrations, and time points over a diel cycle. The expression stability of these genes was evaluated using geNorm and NormFinder algorithms. Although none of these genes exhibited invariable expression levels, cal, tub, rpL17-2 and rpL23 expression levels were the most stable across the different conditions tested. For further validation, these selected genes were used to normalize the expression levels of ribulose-1, 5-bisphosphate carboxylase/oxygenase large unite (HrbcL) over a diel cycle. Results showed that the expression of HrbcL normalized against each of these reference genes was the highest at midday and lowest at midnight, similar to the diel patterns typically documented for this gene in algae. While the validated reference genes will be useful for future gene expression studies on H. akashiwo, we expect that the procedure used in this study may be helpful to future efforts to screen reference genes for other algae.

Metatranscriptome analysis reveals environmental and diel regulation of a Heterosigma akashiwo (raphidophyceae) bloom

“…To examine the effects of low levels of N or P on gene expression in H . akashiwo cultures were treated as previously described (Ji et al ., ), with low N (181.5 μM NaNO 3 and 36.3 μM NaH 2 PO 4 , 5:1 of N:P), low P (883 μM NaNO 3 and 7.06 μM NaH 2 PO 4 , 125:1 of N:P), and nutrient replete ( f /2 medium). Each culture condition was set up in triplicate.…”

Verification of suitable and reliable reference genes for quantitative real-time PCR during adipogenic differentiation in porcine intramuscular stromal-vascular cells

“…In fact, commonly used ICGs can considerably vary between species, tissue types and experimental conditions (Li and Shen, 2013). Furthermore, the conventional use of a single ICG to normalize the target mRNA transcripts may produce unreliable data (Ji et al, 2015).…”

Identification and expression analysis of blue light receptor aureochrome in the harmful alga Heterosigma akashiwo (raphidophyceae)

“…Total RNA from the samples was extracted using the TRIreagent (Molecular Research Center, USA), and purified using Direct-zol RNA Miniprep (Zymo research, USA), as previously reported (Ji et al, 2015). The RNA concentration and quality was determined using NanoDrop spectrophotometer (Thermo, Germany).…”

“…To examine the specificity of each primer, regular PCR were performed and the size of PCR products were examined on agarose gel, and the products were cloned and sequenced. In addition, the PCR amplification efficiency (E) of each primer set was analyzed following E = [10 (À1/slope) ] Â 100% (Supplementary Table S2), in which the slope were obtained from the standard curve generated from a serial dilutions of a pooled cDNA sample, as previously described (Bustin et al, 2009;Coyne et al, 2005;Ji et al, 2015).…”

“…At the end, melting curve analysis was performed to confirm amplification specificity. Ribosomal protein gene rpL17-2 was used as a reference (Ji et al, 2015) with which to normalize Haaureo expression levels. In addition, in this study, our ANOVA analysis showed that the Ct values rpL17-2 was not significantly different among different time point (diel cycle) (Supplementary Table S3).…”