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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.


Figure1. Melting curves for the twelve candidate reference genes. The melting temperature of each amplicon is visualized by plotting the negative derivative of the change in fluorescence divided by the change in temperature in relation to the temperature (−(d/dT) Fluorescence (465–510)).
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ijms-16-12513-f001: Figure1. Melting curves for the twelve candidate reference genes. The melting temperature of each amplicon is visualized by plotting the negative derivative of the change in fluorescence divided by the change in temperature in relation to the temperature (−(d/dT) Fluorescence (465–510)).

Mentions: A total of twelve candidate reference genes (18S, ACT, CYC, EF1α, GAPDH, PLA, RPII, RPS13, TEF2, TUB, UBL and UCE) were selected to normalize the gene expression levels in Sacha inchi using RT-qPCR. The specificity of the primers (Table 1, Supplementary Figure S1) was confirmed by the single peak melting curves of the qPCR products (Figure 1) and the presence of a single band at the correct size for each primer pair in 2% agarose gel electrophoresis (Supplementary Figure S2). The melting temperatures of the PCR products all ranged between 78.74 °C for UCE and 82.95 °C for CYC (Table 1). The amplification efficiencies ranged from 90.55% for RPS13 to 108.93% for CYC, and the correlation coefficients (R2) for the primers all ranged between 0.996 and 0.999 (Table 1).


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)

Figure1. Melting curves for the twelve candidate reference genes. The melting temperature of each amplicon is visualized by plotting the negative derivative of the change in fluorescence divided by the change in temperature in relation to the temperature (−(d/dT) Fluorescence (465–510)).
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-12513-f001: Figure1. Melting curves for the twelve candidate reference genes. The melting temperature of each amplicon is visualized by plotting the negative derivative of the change in fluorescence divided by the change in temperature in relation to the temperature (−(d/dT) Fluorescence (465–510)).
Mentions: A total of twelve candidate reference genes (18S, ACT, CYC, EF1α, GAPDH, PLA, RPII, RPS13, TEF2, TUB, UBL and UCE) were selected to normalize the gene expression levels in Sacha inchi using RT-qPCR. The specificity of the primers (Table 1, Supplementary Figure S1) was confirmed by the single peak melting curves of the qPCR products (Figure 1) and the presence of a single band at the correct size for each primer pair in 2% agarose gel electrophoresis (Supplementary Figure S2). The melting temperatures of the PCR products all ranged between 78.74 °C for UCE and 82.95 °C for CYC (Table 1). The amplification efficiencies ranged from 90.55% for RPS13 to 108.93% for CYC, and the correlation coefficients (R2) for the primers all ranged between 0.996 and 0.999 (Table 1).

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.