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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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Determination of the optimal number of reference genes required for effective normalization.The geNorm program calculated an NF and used the variable V to determine pairwise variation (Vn/Vn+1) between two sequential NFs (NFn and NFn+1). Additional genes are included when V exceeds the cutoff value, which is typically set at 0.15 but is not always achievable. The number of reference genes is deemed optimal when the lowest possible V value is achieved, at which point it is unnecessary to include additional genes in the normalization strategy.
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pone-0112177-g005: Determination of the optimal number of reference genes required for effective normalization.The geNorm program calculated an NF and used the variable V to determine pairwise variation (Vn/Vn+1) between two sequential NFs (NFn and NFn+1). Additional genes are included when V exceeds the cutoff value, which is typically set at 0.15 but is not always achievable. The number of reference genes is deemed optimal when the lowest possible V value is achieved, at which point it is unnecessary to include additional genes in the normalization strategy.

Mentions: Based on the expression stability of the genes and the assumption that two ideal reference genes should not vary with each other under different test conditions [34], geNorm ranked the best out of the three data analysis applications used. geNorm computes the average pair-wise variation of a given candidate reference gene with all the other genes and assigns a score of its expression stability (M) to each gene. Stepwise exclusion of genes with the highest M values (indicating the least stable expressions) before recalculation finally reveals the two most stable candidate genes [35]. After calculating the pair-wise variation Vn/n+1, geNorm selects the optimal number of control genes. The cut-off value is usually set to a default value of 0.15 [34]. Gene expression stability and ranking of 20 candidate reference genes, as calculated by geNorm using nine sets of samples, are presented in Figure 4. Analyses of all fifteen samples revealed that the CYP and EF-1α combination showed the lowest M value (0.31), while 30S RPS20 showed the highest M value (0.89). Among the different organs, GAPDH/30S RPS20, CYP/60S RPL13, and CYP/QCR were the most stably expressed gene combinations in roots, stems, and leaves, respectively; while 18S rRNA, UBQ, and TCTP were the least stably expressed. Among the five developmental stages under study, CYP/60S RPL13, CYP/eIF-5A, aTUB/V-ATP, eIF-5A/SAR1, and aTUB/pol IIa were the most stably expressed combination, respectively, and 30S RPS20 was the least stably expressed gene in all the five stages. Based on these observations, CYP was evidently the most stably expressed gene and may be considered as the most suitable reference gene for the analyses of gene expressions in P. ginseng. Furthermore, the addition of a third reference gene would not have significantly increased the statistical reliability of this calculation, as V2/3 = 0.033 or V3/4 = 0.041 (in roots) was significantly below the default cut-off value of 0.15 (Figure 5). Although the pair-wise variation for all the samples (V2/3) was estimated as 0.145, it was still less than the limiting value. Hence, our study showed that two reference genes were sufficient to normalize gene expression for all the samples of P. ginseng.


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)

Determination of the optimal number of reference genes required for effective normalization.The geNorm program calculated an NF and used the variable V to determine pairwise variation (Vn/Vn+1) between two sequential NFs (NFn and NFn+1). Additional genes are included when V exceeds the cutoff value, which is typically set at 0.15 but is not always achievable. The number of reference genes is deemed optimal when the lowest possible V value is achieved, at which point it is unnecessary to include additional genes in the normalization strategy.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0112177-g005: Determination of the optimal number of reference genes required for effective normalization.The geNorm program calculated an NF and used the variable V to determine pairwise variation (Vn/Vn+1) between two sequential NFs (NFn and NFn+1). Additional genes are included when V exceeds the cutoff value, which is typically set at 0.15 but is not always achievable. The number of reference genes is deemed optimal when the lowest possible V value is achieved, at which point it is unnecessary to include additional genes in the normalization strategy.
Mentions: Based on the expression stability of the genes and the assumption that two ideal reference genes should not vary with each other under different test conditions [34], geNorm ranked the best out of the three data analysis applications used. geNorm computes the average pair-wise variation of a given candidate reference gene with all the other genes and assigns a score of its expression stability (M) to each gene. Stepwise exclusion of genes with the highest M values (indicating the least stable expressions) before recalculation finally reveals the two most stable candidate genes [35]. After calculating the pair-wise variation Vn/n+1, geNorm selects the optimal number of control genes. The cut-off value is usually set to a default value of 0.15 [34]. Gene expression stability and ranking of 20 candidate reference genes, as calculated by geNorm using nine sets of samples, are presented in Figure 4. Analyses of all fifteen samples revealed that the CYP and EF-1α combination showed the lowest M value (0.31), while 30S RPS20 showed the highest M value (0.89). Among the different organs, GAPDH/30S RPS20, CYP/60S RPL13, and CYP/QCR were the most stably expressed gene combinations in roots, stems, and leaves, respectively; while 18S rRNA, UBQ, and TCTP were the least stably expressed. Among the five developmental stages under study, CYP/60S RPL13, CYP/eIF-5A, aTUB/V-ATP, eIF-5A/SAR1, and aTUB/pol IIa were the most stably expressed combination, respectively, and 30S RPS20 was the least stably expressed gene in all the five stages. Based on these observations, CYP was evidently the most stably expressed gene and may be considered as the most suitable reference gene for the analyses of gene expressions in P. ginseng. Furthermore, the addition of a third reference gene would not have significantly increased the statistical reliability of this calculation, as V2/3 = 0.033 or V3/4 = 0.041 (in roots) was significantly below the default cut-off value of 0.15 (Figure 5). Although the pair-wise variation for all the samples (V2/3) was estimated as 0.145, it was still less than the limiting value. Hence, our study showed that two reference genes were sufficient to normalize gene expression for all the samples of P. ginseng.

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