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Selection of reference genes for quantitative real-time PCR normalization in Panax ginseng at different stages of growth and in different organs.

Liu J, Wang Q, Sun M, Zhu L, Yang M, Zhao Y - PLoS ONE (2014)

Bottom Line: Although the best reference genes were found to vary across different samples, CYP and EF-1α were the most stable genes amongst all samples.CYP/60S RPL13, CYP/eIF-5A, aTUB/V-ATP, eIF-5A/SAR1, and aTUB/pol IIa were the most stably expressed combinations in each of the five developmental stages.Our study serves as a foundation for developing an accurate method of qRT-PCR and will benefit future studies on gene expression profiles of Panax Ginseng.

View Article: PubMed Central - PubMed

Affiliation: Traditional Chinese Medicine and Biotechnology Research and Development Center, Changchun University of Traditional Chinese Medicine, Changchun, Jilin, People's Republic of China.

ABSTRACT
Quantitative real-time reverse transcription PCR (qRT-PCR) has become a widely used method for gene expression analysis; however, its data interpretation largely depends on the stability of reference genes. The transcriptomics of Panax ginseng, one of the most popular and traditional ingredients used in Chinese medicines, is increasingly being studied. Furthermore, it is vital to establish a series of reliable reference genes when qRT-PCR is used to assess the gene expression profile of ginseng. In this study, we screened out candidate reference genes for ginseng using gene expression data generated by a high-throughput sequencing platform. Based on the statistical tests, 20 reference genes (10 traditional housekeeping genes and 10 novel genes) were selected. These genes were tested for the normalization of expression levels in five growth stages and three distinct plant organs of ginseng by qPCR. These genes were subsequently ranked and compared according to the stability of their expressions using geNorm, NormFinder, and BestKeeper computational programs. Although the best reference genes were found to vary across different samples, CYP and EF-1α were the most stable genes amongst all samples. GAPDH/30S RPS20, CYP/60S RPL13 and CYP/QCR were the optimum pair of reference genes in the roots, stems, and leaves. CYP/60S RPL13, CYP/eIF-5A, aTUB/V-ATP, eIF-5A/SAR1, and aTUB/pol IIa were the most stably expressed combinations in each of the five developmental stages. Our study serves as a foundation for developing an accurate method of qRT-PCR and will benefit future studies on gene expression profiles of Panax Ginseng.

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Specificity of primer pairs for RT-qPCR amplification.Agarose gel (2%) electrophoresis showing amplification of a specific PCR product of the expected size for each gene (M:DL1000 DNA Marker).
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pone-0112177-g002: Specificity of primer pairs for RT-qPCR amplification.Agarose gel (2%) electrophoresis showing amplification of a specific PCR product of the expected size for each gene (M:DL1000 DNA Marker).

Mentions: By reverse transcription PCR, the specificity of the primers used for candidate reference genes was verified. A single band for each gene was revealed through electrophoresis, without primer-dimers or non-specific amplification (Figure 2).


Selection of reference genes for quantitative real-time PCR normalization in Panax ginseng at different stages of growth and in different organs.

Liu J, Wang Q, Sun M, Zhu L, Yang M, Zhao Y - PLoS ONE (2014)

Specificity of primer pairs for RT-qPCR amplification.Agarose gel (2%) electrophoresis showing amplification of a specific PCR product of the expected size for each gene (M:DL1000 DNA Marker).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4230945&req=5

pone-0112177-g002: Specificity of primer pairs for RT-qPCR amplification.Agarose gel (2%) electrophoresis showing amplification of a specific PCR product of the expected size for each gene (M:DL1000 DNA Marker).
Mentions: By reverse transcription PCR, the specificity of the primers used for candidate reference genes was verified. A single band for each gene was revealed through electrophoresis, without primer-dimers or non-specific amplification (Figure 2).

Bottom Line: Although the best reference genes were found to vary across different samples, CYP and EF-1α were the most stable genes amongst all samples.CYP/60S RPL13, CYP/eIF-5A, aTUB/V-ATP, eIF-5A/SAR1, and aTUB/pol IIa were the most stably expressed combinations in each of the five developmental stages.Our study serves as a foundation for developing an accurate method of qRT-PCR and will benefit future studies on gene expression profiles of Panax Ginseng.

View Article: PubMed Central - PubMed

Affiliation: Traditional Chinese Medicine and Biotechnology Research and Development Center, Changchun University of Traditional Chinese Medicine, Changchun, Jilin, People's Republic of China.

ABSTRACT
Quantitative real-time reverse transcription PCR (qRT-PCR) has become a widely used method for gene expression analysis; however, its data interpretation largely depends on the stability of reference genes. The transcriptomics of Panax ginseng, one of the most popular and traditional ingredients used in Chinese medicines, is increasingly being studied. Furthermore, it is vital to establish a series of reliable reference genes when qRT-PCR is used to assess the gene expression profile of ginseng. In this study, we screened out candidate reference genes for ginseng using gene expression data generated by a high-throughput sequencing platform. Based on the statistical tests, 20 reference genes (10 traditional housekeeping genes and 10 novel genes) were selected. These genes were tested for the normalization of expression levels in five growth stages and three distinct plant organs of ginseng by qPCR. These genes were subsequently ranked and compared according to the stability of their expressions using geNorm, NormFinder, and BestKeeper computational programs. Although the best reference genes were found to vary across different samples, CYP and EF-1α were the most stable genes amongst all samples. GAPDH/30S RPS20, CYP/60S RPL13 and CYP/QCR were the optimum pair of reference genes in the roots, stems, and leaves. CYP/60S RPL13, CYP/eIF-5A, aTUB/V-ATP, eIF-5A/SAR1, and aTUB/pol IIa were the most stably expressed combinations in each of the five developmental stages. Our study serves as a foundation for developing an accurate method of qRT-PCR and will benefit future studies on gene expression profiles of Panax Ginseng.

Show MeSH