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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-1Ī± 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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RT-qPCR CT values for the candidate reference genes (nā€Š=ā€Š3).Expression date displayed as CT values for each reference gene in all ginseng samples. A line across the box is depicted as the median. The box indicates the 25th and 75th percentiles. Whiskers represent the maximum and minimum values.
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pone-0112177-g003: RT-qPCR CT values for the candidate reference genes (nā€Š=ā€Š3).Expression date displayed as CT values for each reference gene in all ginseng samples. A line across the box is depicted as the median. The box indicates the 25th and 75th percentiles. Whiskers represent the maximum and minimum values.

Mentions: With a higher gene expression, a smaller Ct value was obtained, and vice versa. Figure 3 shows a relatively broad range of Ct values for all the 20 putative reference genes. The highest Ct value was 26.40 (bTUB), while the lowest was 15.06 (18S rRNA). Ct values of the remaining genes were distributed between 19 and 24. On comparing the Ct values of the 20 candidate reference genes, the expression level of each reference gene was found to differ, with respect to the developmental stage or the organ under study. The expression patterns of the 20 reference genes displayed irregular variation; this may be attributed to change in the level of reference gene expression abundance with the cell type and the developmental stage [33]. Therefore, successful gene expression analysis under different experimental conditions in ginseng requires careful selection of reliable reference genes.


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)

RT-qPCR CT values for the candidate reference genes (nā€Š=ā€Š3).Expression date displayed as CT values for each reference gene in all ginseng samples. A line across the box is depicted as the median. The box indicates the 25th and 75th percentiles. Whiskers represent the maximum and minimum values.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112177-g003: RT-qPCR CT values for the candidate reference genes (nā€Š=ā€Š3).Expression date displayed as CT values for each reference gene in all ginseng samples. A line across the box is depicted as the median. The box indicates the 25th and 75th percentiles. Whiskers represent the maximum and minimum values.
Mentions: With a higher gene expression, a smaller Ct value was obtained, and vice versa. Figure 3 shows a relatively broad range of Ct values for all the 20 putative reference genes. The highest Ct value was 26.40 (bTUB), while the lowest was 15.06 (18S rRNA). Ct values of the remaining genes were distributed between 19 and 24. On comparing the Ct values of the 20 candidate reference genes, the expression level of each reference gene was found to differ, with respect to the developmental stage or the organ under study. The expression patterns of the 20 reference genes displayed irregular variation; this may be attributed to change in the level of reference gene expression abundance with the cell type and the developmental stage [33]. Therefore, successful gene expression analysis under different experimental conditions in ginseng requires careful selection of reliable reference genes.

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-1Ī± 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