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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.


Relative quantification of the AGAMOUS homolog (PvoAG) in Sacha inchi using the validated reference genes for normalization. (A) PvoAG expression pattern in adult plants. The four kinds of bars indicate PvoAG expression levels normalized by different reference genes RPS13, CYC, GAPDH and 18S respectively; and (B) PvoAG expression pattern during flower development. The four kinds of bars indicate PvoAG expression levels normalized by different reference genes PLA, ACT, RPS13 and TUB respectively. YL, young leaf; R, root; S, stem; ML, mature leaf; YI, young inflorescence; SD, seed (90 DAP); IB, inflorescence bud; FF, female flower; MF, male flower.
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ijms-16-12513-f003: Relative quantification of the AGAMOUS homolog (PvoAG) in Sacha inchi using the validated reference genes for normalization. (A) PvoAG expression pattern in adult plants. The four kinds of bars indicate PvoAG expression levels normalized by different reference genes RPS13, CYC, GAPDH and 18S respectively; and (B) PvoAG expression pattern during flower development. The four kinds of bars indicate PvoAG expression levels normalized by different reference genes PLA, ACT, RPS13 and TUB respectively. YL, young leaf; R, root; S, stem; ML, mature leaf; YI, young inflorescence; SD, seed (90 DAP); IB, inflorescence bud; FF, female flower; MF, male flower.

Mentions: Sacha inchi AGAMOUS (PvoAG, GenBank GADC01013770), with homologs in other plants that are mainly expressed in floral organs [29,30,31], was chosen to further validate the reliability of the selected reference genes for the normalization of RT-qPCR data in Sacha inchi adult plants and flower developmental stages. The most stable reference genes identified for adult plants (RPS13 and CYC) and during flower development (PLA and ACT) were used as internal controls for data normalization. For comparison, the least stable reference genes identified in adult plants (GAPDH and 18S) and during flower development (RPS13 and TUB) were also considered. The results demonstrated that the expression patterns of PvoAG differed when using the most and least stable reference genes for normalization (Figure 3). In adult plants (Figure 3A), when the RPS13 and CYC genes were used for normalization, PvoAG was predominantly expressed in young inflorescences with relatively lower expression in seeds (90 DAP). However, the expression level of PvoAG in seeds (90 DAP) was substantially greater than in young inflorescences when using the least stable reference genes (GAPDH and 18S) for normalization. The PvoAG gene was also found to be expressed in mature roots when using GAPDH for normalization. During flower development (Figure 3B), when PLA and ACT were used for normalization, PvoAG was expressed in all developmental stages and at a higher level in male flowers. When RPS13 and TUB were considered, the expression pattern of PvoAG was similar to that obtained when using the most stable reference genes, but the expression level was over-estimated in male flowers. These findings suggest that the choice of reliable reference genes is essential for the accurate normalization of target gene expression levels.


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)

Relative quantification of the AGAMOUS homolog (PvoAG) in Sacha inchi using the validated reference genes for normalization. (A) PvoAG expression pattern in adult plants. The four kinds of bars indicate PvoAG expression levels normalized by different reference genes RPS13, CYC, GAPDH and 18S respectively; and (B) PvoAG expression pattern during flower development. The four kinds of bars indicate PvoAG expression levels normalized by different reference genes PLA, ACT, RPS13 and TUB respectively. YL, young leaf; R, root; S, stem; ML, mature leaf; YI, young inflorescence; SD, seed (90 DAP); IB, inflorescence bud; FF, female flower; MF, male flower.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-12513-f003: Relative quantification of the AGAMOUS homolog (PvoAG) in Sacha inchi using the validated reference genes for normalization. (A) PvoAG expression pattern in adult plants. The four kinds of bars indicate PvoAG expression levels normalized by different reference genes RPS13, CYC, GAPDH and 18S respectively; and (B) PvoAG expression pattern during flower development. The four kinds of bars indicate PvoAG expression levels normalized by different reference genes PLA, ACT, RPS13 and TUB respectively. YL, young leaf; R, root; S, stem; ML, mature leaf; YI, young inflorescence; SD, seed (90 DAP); IB, inflorescence bud; FF, female flower; MF, male flower.
Mentions: Sacha inchi AGAMOUS (PvoAG, GenBank GADC01013770), with homologs in other plants that are mainly expressed in floral organs [29,30,31], was chosen to further validate the reliability of the selected reference genes for the normalization of RT-qPCR data in Sacha inchi adult plants and flower developmental stages. The most stable reference genes identified for adult plants (RPS13 and CYC) and during flower development (PLA and ACT) were used as internal controls for data normalization. For comparison, the least stable reference genes identified in adult plants (GAPDH and 18S) and during flower development (RPS13 and TUB) were also considered. The results demonstrated that the expression patterns of PvoAG differed when using the most and least stable reference genes for normalization (Figure 3). In adult plants (Figure 3A), when the RPS13 and CYC genes were used for normalization, PvoAG was predominantly expressed in young inflorescences with relatively lower expression in seeds (90 DAP). However, the expression level of PvoAG in seeds (90 DAP) was substantially greater than in young inflorescences when using the least stable reference genes (GAPDH and 18S) for normalization. The PvoAG gene was also found to be expressed in mature roots when using GAPDH for normalization. During flower development (Figure 3B), when PLA and ACT were used for normalization, PvoAG was expressed in all developmental stages and at a higher level in male flowers. When RPS13 and TUB were considered, the expression pattern of PvoAG was similar to that obtained when using the most stable reference genes, but the expression level was over-estimated in male flowers. These findings suggest that the choice of reliable reference genes is essential for the accurate normalization of target gene expression levels.

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.