Dataset Information

Selection and Validation of Reference Genes for mRNA Expression by Quantitative Real-Time PCR Analysis in Neolamarckia cadamba.

ABSTRACT: Neolamarckia cadamba is an economically-important fast-growing tree species in South China and Southeast Asia. As a prerequisite first step for future gene expression studies, we have identified and characterized a series of stable reference genes that can be used as controls for quantitative real time PCR (qRT-PCR) expression analysis in this study. The expression stability of 15 candidate reference genes in various tissues and mature leaves under different conditions was evaluated using four different algorithms, i.e., geNorm, NormFinder, BestKeeper and RefFinder. Our results showed that SAMDC was the most stable of the selected reference genes across the set of all samples, mature leaves at different photosynthetic cycles and under drought stress, whereas RPL10A had the most stable expression in various tissues. PGK and RPS25 were considered the most suitable reference for mature leaves at different developmental stages and under cold treatment, respectively. Additionally, the gene expression profiles of sucrose transporter 4 (NcSUT4), and 9-cis-epoxycarotenoid dioxygenase 3 (NcNCED3) were used to confirm the validity of candidate reference genes. Collectively, our study is the first report to validate the optimal reference genes for normalization under various conditions in N. cadamba and will benefit the future discovery of gene function in this species.

SUBMITTER: Huang T

PROVIDER: S-EPMC6006177 | biostudies-literature | 2018 Jun

REPOSITORIES: biostudies-literature

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Selection and Validation of Reference Genes for mRNA Expression by Quantitative Real-Time PCR Analysis in Neolamarckia cadamba.

Huang Tian T Long Jianmei J Liu Si-Wen SW Yang Zi-Wei ZW Zhu Qi-Jin QJ Zhao Xiao-Lan XL Peng Changcao C

Scientific reports 20180618 1

Neolamarckia cadamba is an economically-important fast-growing tree species in South China and Southeast Asia. As a prerequisite first step for future gene expression studies, we have identified and characterized a series of stable reference genes that can be used as controls for quantitative real time PCR (qRT-PCR) expression analysis in this study. The expression stability of 15 candidate reference genes in various tissues and mature leaves under different conditions was evaluated using four d ...[more]

PMID: 29915368

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Project description:Lycoris aurea (L' Hér.) Herb, a perennial grass species, produces a unique variety of pharmacologically active Amaryllidaceae alkaloids. However, the key enzymes and their expression pattern involved in the biosynthesis of Amaryllidaceae alkaloids (especially for galanthamine) are far from being fully understood. Quantitative real-time polymerase chain reaction (qRT-PCR), a commonly used method for quantifying gene expression, requires stable reference genes to normalize its data. In this study, to choose the appropriate reference genes under different experimental conditions, 14 genes including YLS8 (mitosis protein YLS8), CYP2 (Cyclophilin 2), CYP 1 (Cyclophilin 1), TIP41 (TIP41-like protein), EXP2 (Expressed protein 2), PTBP1 (Polypyrimidine tract-binding protein 1), EXP1 (Expressed protein 1), PP2A (Serine/threonine-protein phosphatase 2A), ?-TUB (?-tubulin), ?-TUB (?-tubulin), EF1-? (Elongation factor 1-?), UBC (Ubiquitin-conjugating enzyme), ACT (Actin) and GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) were selected from the transcriptome datasets of L. aurea. And then, expressions of these genes were assessed by qRT-PCR in various tissues and the roots under different treatments. The expression stability of the 14 candidates was analyzed by three commonly used software programs (geNorm, NormFinder, and BestKeeper), and their results were further integrated into a comprehensive ranking based on the geometric mean. The results show the relatively stable genes for each subset as follows: (1) EXP1 and TIP41 for all samples; (2) UBC and EXP1 for NaCl stress; (3) PTBP1 and EXP1 for heat stress, polyethylene glycol (PEG) stress and ABA treatment; (4) UBC and CYP2 for cold stress; (5) PTBP1 and PP2A for sodium nitroprusside (SNP) treatment; (6) CYP1 and TIP41 for methyl jasmonate (MeJA) treatment; and (7) EXP1 and TIP41 for various tissues. The reliability of these results was further enhanced through comparison between part qRT-PCR result and RNA sequencing (RNA-seq) data. In summary, our results identified appropriate reference genes for qRT-PCR in L. aurea, and will facilitate gene expression studies under these conditions.

Dataset Information

Selection and Validation of Reference Genes for mRNA Expression by Quantitative Real-Time PCR Analysis in Neolamarckia cadamba.

Publications

Selection and Validation of Reference Genes for mRNA Expression by Quantitative Real-Time PCR Analysis in Neolamarckia cadamba.

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