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Stable Reference Gene Selection for RT-qPCR Analysis in Synechococcus elongatus PCC 7942 under Abiotic Stresses

Synechococcus elongatus PCC 7942 (S. elongatus PCC 7942) is a model cyanobacteria species for circadian clock mechanism studies. It has also been widely used as a bioreactor to produce biofuels and other metabolic products. Quantitative real-time PCR (qPCR) technology is the most commonly used metho...

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Autores principales: Luo, Xiao, Li, Jiaxing, Chang, Tianliang, He, Hongyan, Zhao, Yi, Yang, Xue, Zhao, Yuwei, Xu, Yao
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6500708/
https://www.ncbi.nlm.nih.gov/pubmed/31139651
http://dx.doi.org/10.1155/2019/7630601
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author Luo, Xiao
Li, Jiaxing
Chang, Tianliang
He, Hongyan
Zhao, Yi
Yang, Xue
Zhao, Yuwei
Xu, Yao
author_facet Luo, Xiao
Li, Jiaxing
Chang, Tianliang
He, Hongyan
Zhao, Yi
Yang, Xue
Zhao, Yuwei
Xu, Yao
author_sort Luo, Xiao
collection PubMed
description Synechococcus elongatus PCC 7942 (S. elongatus PCC 7942) is a model cyanobacteria species for circadian clock mechanism studies. It has also been widely used as a bioreactor to produce biofuels and other metabolic products. Quantitative real-time PCR (qPCR) technology is the most commonly used method for studying the expression of specific genes, in which the relative expression level of target genes is calibrated by stably expressed internal reference genes. In this work, we examined the expression of nine candidate reference genes in time-course samples of S. elongatus PCC 7942 under no treatment (control), NaCl-stress conditions, H(2)O(2)-stress conditions, and high light-stress conditions. Based on the qPCR amplification parameters, the stability ranking of these candidate reference genes was established by three statistical software programs, geNorm, NormFinder, and BestKeeper. Considering all the stress conditions or high light stress alone, the results showed that the combination of prs and secA was the best choice for the double reference gene calibration method by qPCR. The combination of secA and ppc, rimM and rnpA, rnpA, and ilvD was most stable under no treatment, NaCl-stress conditions, and H(2)O(2)-stress conditions, respectively. rimM was stable under only special conditions and should be carefully chosen. 16S and rnpB were not suitable as internal reference genes for S. elongatus PCC 7942 qPCR experiments under all experimental conditions. To validate the above results, a cyanobacterial core clock gene, kaiC, was used to evaluate the actual performance of the optimized reference genes by qPCR, as well as the worst reference genes under different stress conditions. The results indicated that the best reference gene yielded more accurate calibration results for qPCR experiments carried out in S. elongatus PCC 7942 time-course samples.
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spelling pubmed-65007082019-05-28 Stable Reference Gene Selection for RT-qPCR Analysis in Synechococcus elongatus PCC 7942 under Abiotic Stresses Luo, Xiao Li, Jiaxing Chang, Tianliang He, Hongyan Zhao, Yi Yang, Xue Zhao, Yuwei Xu, Yao Biomed Res Int Research Article Synechococcus elongatus PCC 7942 (S. elongatus PCC 7942) is a model cyanobacteria species for circadian clock mechanism studies. It has also been widely used as a bioreactor to produce biofuels and other metabolic products. Quantitative real-time PCR (qPCR) technology is the most commonly used method for studying the expression of specific genes, in which the relative expression level of target genes is calibrated by stably expressed internal reference genes. In this work, we examined the expression of nine candidate reference genes in time-course samples of S. elongatus PCC 7942 under no treatment (control), NaCl-stress conditions, H(2)O(2)-stress conditions, and high light-stress conditions. Based on the qPCR amplification parameters, the stability ranking of these candidate reference genes was established by three statistical software programs, geNorm, NormFinder, and BestKeeper. Considering all the stress conditions or high light stress alone, the results showed that the combination of prs and secA was the best choice for the double reference gene calibration method by qPCR. The combination of secA and ppc, rimM and rnpA, rnpA, and ilvD was most stable under no treatment, NaCl-stress conditions, and H(2)O(2)-stress conditions, respectively. rimM was stable under only special conditions and should be carefully chosen. 16S and rnpB were not suitable as internal reference genes for S. elongatus PCC 7942 qPCR experiments under all experimental conditions. To validate the above results, a cyanobacterial core clock gene, kaiC, was used to evaluate the actual performance of the optimized reference genes by qPCR, as well as the worst reference genes under different stress conditions. The results indicated that the best reference gene yielded more accurate calibration results for qPCR experiments carried out in S. elongatus PCC 7942 time-course samples. Hindawi 2019-04-21 /pmc/articles/PMC6500708/ /pubmed/31139651 http://dx.doi.org/10.1155/2019/7630601 Text en Copyright © 2019 Xiao Luo et al. https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Research Article
Luo, Xiao
Li, Jiaxing
Chang, Tianliang
He, Hongyan
Zhao, Yi
Yang, Xue
Zhao, Yuwei
Xu, Yao
Stable Reference Gene Selection for RT-qPCR Analysis in Synechococcus elongatus PCC 7942 under Abiotic Stresses
title Stable Reference Gene Selection for RT-qPCR Analysis in Synechococcus elongatus PCC 7942 under Abiotic Stresses
title_full Stable Reference Gene Selection for RT-qPCR Analysis in Synechococcus elongatus PCC 7942 under Abiotic Stresses
title_fullStr Stable Reference Gene Selection for RT-qPCR Analysis in Synechococcus elongatus PCC 7942 under Abiotic Stresses
title_full_unstemmed Stable Reference Gene Selection for RT-qPCR Analysis in Synechococcus elongatus PCC 7942 under Abiotic Stresses
title_short Stable Reference Gene Selection for RT-qPCR Analysis in Synechococcus elongatus PCC 7942 under Abiotic Stresses
title_sort stable reference gene selection for rt-qpcr analysis in synechococcus elongatus pcc 7942 under abiotic stresses
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6500708/
https://www.ncbi.nlm.nih.gov/pubmed/31139651
http://dx.doi.org/10.1155/2019/7630601
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