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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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RPKM value distribution of 20 candidate reference genes.LP, leaf-expansion period; FS, the flower stage; GFS, the green fruit stage; RFS, the red fruit stages; RGS, the root growing after fruit stage.
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pone-0112177-g001: RPKM value distribution of 20 candidate reference genes.LP, leaf-expansion period; FS, the flower stage; GFS, the green fruit stage; RFS, the red fruit stages; RGS, the root growing after fruit stage.

Mentions: In the present study, we screened ten housekeeping genes (ACT1, GAPDH, 18SrRNA, UBQ, aTUB, bTUB, CYP, eIF-5A, F-box, and EF-1α). Besides 18S rRNA, the RPKM value distribution of the remaining nine housekeeping genes was in the range of 90–500. According to this observation, the RPKM value selection range of the candidate reference genes was expanded to 50–500. To evaluate the gene expression volatility, we examined the variability in PRKM values among the 15 databases. The CV and MFC values of the ten traditional housekeeping genes were calculated in one organ during the five stages of growth or in the three vegetative organs at one growth stage (Table 1-a). The CV values of the housekeeping genes were found to vary from 3.06% to 88.21%, while the MFC values ranged from 1.06 to 8.76. In order to screen more stable reference genes, we set the threshold values for CV to <20% and MFC to <1.5. Additionally, ten novel genes (CDP, 6-PG, 30S RPS20, 60S RPL13, V-ATP, pol IIa, ARF, QCR, SAR1, and TCTP) were screened as candidate reference genes (Table 1-b). Screening of the potential reference genes was based on the statistical tests (CV and MFC), which reflected the RPKM values of stably and moderately or highly expressed genes among all the databases. RPKM value expression abundance ratios are presented in Figure 1. To determine the distribution of transcript populations of 20 candidate reference genes in three vegetative organs of ginseng during the five stages of growth, the quantity of transcript for each gene was estimated as a ratio relative to the sum of the 20 transcript populations. The results clearly revealed a fluctuation in the relative magnitude of RPKM values and the ratios, thus indicating that all of the 20 genes did not exhibit stable expression patterns. A summary of the sequence information for the 20 ginseng candidate reference genes is presented in Table 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)

RPKM value distribution of 20 candidate reference genes.LP, leaf-expansion period; FS, the flower stage; GFS, the green fruit stage; RFS, the red fruit stages; RGS, the root growing after fruit stage.
© Copyright Policy
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC4230945&req=5

pone-0112177-g001: RPKM value distribution of 20 candidate reference genes.LP, leaf-expansion period; FS, the flower stage; GFS, the green fruit stage; RFS, the red fruit stages; RGS, the root growing after fruit stage.
Mentions: In the present study, we screened ten housekeeping genes (ACT1, GAPDH, 18SrRNA, UBQ, aTUB, bTUB, CYP, eIF-5A, F-box, and EF-1α). Besides 18S rRNA, the RPKM value distribution of the remaining nine housekeeping genes was in the range of 90–500. According to this observation, the RPKM value selection range of the candidate reference genes was expanded to 50–500. To evaluate the gene expression volatility, we examined the variability in PRKM values among the 15 databases. The CV and MFC values of the ten traditional housekeeping genes were calculated in one organ during the five stages of growth or in the three vegetative organs at one growth stage (Table 1-a). The CV values of the housekeeping genes were found to vary from 3.06% to 88.21%, while the MFC values ranged from 1.06 to 8.76. In order to screen more stable reference genes, we set the threshold values for CV to <20% and MFC to <1.5. Additionally, ten novel genes (CDP, 6-PG, 30S RPS20, 60S RPL13, V-ATP, pol IIa, ARF, QCR, SAR1, and TCTP) were screened as candidate reference genes (Table 1-b). Screening of the potential reference genes was based on the statistical tests (CV and MFC), which reflected the RPKM values of stably and moderately or highly expressed genes among all the databases. RPKM value expression abundance ratios are presented in Figure 1. To determine the distribution of transcript populations of 20 candidate reference genes in three vegetative organs of ginseng during the five stages of growth, the quantity of transcript for each gene was estimated as a ratio relative to the sum of the 20 transcript populations. The results clearly revealed a fluctuation in the relative magnitude of RPKM values and the ratios, thus indicating that all of the 20 genes did not exhibit stable expression patterns. A summary of the sequence information for the 20 ginseng candidate reference genes is presented in Table 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