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

Ji N, Li L, Lin L, Lin S - PLoS ONE (2015)

Bottom Line: The raphidophyte Heterosigma akashiwo is a globally distributed harmful alga that has been associated with fish kills in coastal waters.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, Fujian 361005, China.

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.

No MeSH data available.


Gene expression stability and ranking of the candidate reference genes as calculated by geNorm.Average expression stability value (M) was calculated by stepwise exclusion of the least stable gene compared across different temperatures (Temperature), different light intensities (Light), different nutrient (N: P) ratios (Nutrient), different time points in the diel cycle (Diel), and the combination of all the samples (Total). In each plot, the least stable gene is on the left, and the most stable gene on the right.
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pone.0132183.g002: Gene expression stability and ranking of the candidate reference genes as calculated by geNorm.Average expression stability value (M) was calculated by stepwise exclusion of the least stable gene compared across different temperatures (Temperature), different light intensities (Light), different nutrient (N: P) ratios (Nutrient), different time points in the diel cycle (Diel), and the combination of all the samples (Total). In each plot, the least stable gene is on the left, and the most stable gene on the right.

Mentions: Average expression stability values (M) were obtained using geNorm, and all candidate genes were ranked based on the M values (Fig 2). All ten genes investigated in this study showed M values below the threshold value 1.5, indicating that the expression levels of these genes were relatively stable under all the conditions we examined. By comparison, as shown in Fig 2, the most stable genes were cal and tub under temperature treatment, 18S and tub under light treatment, rpL17-2 and rpL23 in the diel cycle samples, cal and rpL17-2 under nutrient treatment and all treatments combined (M = 0.488) (Total). We found that gapdh was the least stably expressed under temperature treatment, light treatment, time points over the diel cycle, and total (M = 1.219), and ef1 was the least stably expressed gene under nutrient treatment. In addition, our ANONA analysis showed that the variations in the gapdh and ef1 Ct values were attributable to experimental treatments rather than inconsistencies between replicates, because the between group variance was much higher than the within group variance (S3 Table).


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

Ji N, Li L, Lin L, Lin S - PLoS ONE (2015)

Gene expression stability and ranking of the candidate reference genes as calculated by geNorm.Average expression stability value (M) was calculated by stepwise exclusion of the least stable gene compared across different temperatures (Temperature), different light intensities (Light), different nutrient (N: P) ratios (Nutrient), different time points in the diel cycle (Diel), and the combination of all the samples (Total). In each plot, the least stable gene is on the left, and the most stable gene on the right.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4489630&req=5

pone.0132183.g002: Gene expression stability and ranking of the candidate reference genes as calculated by geNorm.Average expression stability value (M) was calculated by stepwise exclusion of the least stable gene compared across different temperatures (Temperature), different light intensities (Light), different nutrient (N: P) ratios (Nutrient), different time points in the diel cycle (Diel), and the combination of all the samples (Total). In each plot, the least stable gene is on the left, and the most stable gene on the right.
Mentions: Average expression stability values (M) were obtained using geNorm, and all candidate genes were ranked based on the M values (Fig 2). All ten genes investigated in this study showed M values below the threshold value 1.5, indicating that the expression levels of these genes were relatively stable under all the conditions we examined. By comparison, as shown in Fig 2, the most stable genes were cal and tub under temperature treatment, 18S and tub under light treatment, rpL17-2 and rpL23 in the diel cycle samples, cal and rpL17-2 under nutrient treatment and all treatments combined (M = 0.488) (Total). We found that gapdh was the least stably expressed under temperature treatment, light treatment, time points over the diel cycle, and total (M = 1.219), and ef1 was the least stably expressed gene under nutrient treatment. In addition, our ANONA analysis showed that the variations in the gapdh and ef1 Ct values were attributable to experimental treatments rather than inconsistencies between replicates, because the between group variance was much higher than the within group variance (S3 Table).

Bottom Line: The raphidophyte Heterosigma akashiwo is a globally distributed harmful alga that has been associated with fish kills in coastal waters.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.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Marine Environmental Science, Xiamen University, Xiamen, Fujian 361005, China.

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.

No MeSH data available.