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Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR.

Niu L, Tao YB, Chen MS, Fu Q, Li C, Dong Y, Wang X, He H, Xu ZF - Int J Mol Sci (2015)

Bottom Line: The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings.Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization.Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Science and Technology of China, Hefei 230027, China. niulongjian@126.com.

ABSTRACT
Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.

No MeSH data available.


Average cycle threshold (Ct) values for the twelve candidate reference genes. Boxes indicate the interquartile range. Lines across the boxes indicate the average Ct values. Whiskers represent 95% confidence intervals, and black dots represent outliers.
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ijms-16-12513-f002: Average cycle threshold (Ct) values for the twelve candidate reference genes. Boxes indicate the interquartile range. Lines across the boxes indicate the average Ct values. Whiskers represent 95% confidence intervals, and black dots represent outliers.

Mentions: The transcript levels of the twelve candidate reference genes, presented as the cycle threshold (Ct) values, were obtained by RT-qPCR. The box-plot analysis was performed using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). The data used to produce the box-plot were shown in the supplementary Table S1. The results indicated that the candidate reference genes evaluated in this study encompassed a wide range of Ct values, ranging from 8 to 30, with the majority ranging from 16 to 27 (Figure 2, Supplementary Table S1). The 18S gene was the most abundant reference gene in the tested Sacha inchi tissues with the lowest mean Ct value of 9, whereas RPS13 was the least abundant reference gene with the highest mean Ct value of 25. The results also revealed that the PLA gene was characterized by the smallest variation in transcript levels among plant tissues, whereas the GAPDH gene displayed the largest variation among tissues.


Selection of Reliable Reference Genes for Gene Expression Studies of a Promising Oilseed Crop, Plukenetia volubilis, by Real-Time Quantitative PCR.

Niu L, Tao YB, Chen MS, Fu Q, Li C, Dong Y, Wang X, He H, Xu ZF - Int J Mol Sci (2015)

Average cycle threshold (Ct) values for the twelve candidate reference genes. Boxes indicate the interquartile range. Lines across the boxes indicate the average Ct values. Whiskers represent 95% confidence intervals, and black dots represent outliers.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4490458&req=5

ijms-16-12513-f002: Average cycle threshold (Ct) values for the twelve candidate reference genes. Boxes indicate the interquartile range. Lines across the boxes indicate the average Ct values. Whiskers represent 95% confidence intervals, and black dots represent outliers.
Mentions: The transcript levels of the twelve candidate reference genes, presented as the cycle threshold (Ct) values, were obtained by RT-qPCR. The box-plot analysis was performed using GraphPad Prism 5 software (GraphPad Software, San Diego, CA, USA). The data used to produce the box-plot were shown in the supplementary Table S1. The results indicated that the candidate reference genes evaluated in this study encompassed a wide range of Ct values, ranging from 8 to 30, with the majority ranging from 16 to 27 (Figure 2, Supplementary Table S1). The 18S gene was the most abundant reference gene in the tested Sacha inchi tissues with the lowest mean Ct value of 9, whereas RPS13 was the least abundant reference gene with the highest mean Ct value of 25. The results also revealed that the PLA gene was characterized by the smallest variation in transcript levels among plant tissues, whereas the GAPDH gene displayed the largest variation among tissues.

Bottom Line: The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings.Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization.Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.

View Article: PubMed Central - PubMed

Affiliation: School of Life Sciences, University of Science and Technology of China, Hefei 230027, China. niulongjian@126.com.

ABSTRACT
Real-time quantitative PCR (RT-qPCR) is a reliable and widely used method for gene expression analysis. The accuracy of the determination of a target gene expression level by RT-qPCR demands the use of appropriate reference genes to normalize the mRNA levels among different samples. However, suitable reference genes for RT-qPCR have not been identified in Sacha inchi (Plukenetia volubilis), a promising oilseed crop known for its polyunsaturated fatty acid (PUFA)-rich seeds. In this study, using RT-qPCR, twelve candidate reference genes were examined in seedlings and adult plants, during flower and seed development and for the entire growth cycle of Sacha inchi. Four statistical algorithms (delta cycle threshold (ΔCt), BestKeeper, geNorm, and NormFinder) were used to assess the expression stabilities of the candidate genes. The results showed that ubiquitin-conjugating enzyme (UCE), actin (ACT) and phospholipase A22 (PLA) were the most stable genes in Sacha inchi seedlings. For roots, stems, leaves, flowers, and seeds from adult plants, 30S ribosomal protein S13 (RPS13), cyclophilin (CYC) and elongation factor-1alpha (EF1α) were recommended as reference genes for RT-qPCR. During the development of reproductive organs, PLA, ACT and UCE were the optimal reference genes for flower development, whereas UCE, RPS13 and RNA polymerase II subunit (RPII) were optimal for seed development. Considering the entire growth cycle of Sacha inchi, UCE, ACT and EF1α were sufficient for the purpose of normalization. Our results provide useful guidelines for the selection of reliable reference genes for the normalization of RT-qPCR data for seedlings and adult plants, for reproductive organs, and for the entire growth cycle of Sacha inchi.

No MeSH data available.