Dataset Information

Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Lycoris aurea.

ABSTRACT: 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.

SUBMITTER: Ma R

PROVIDER: S-EPMC4843812 | biostudies-other | 2016

REPOSITORIES: biostudies-other

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Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Lycoris aurea.

Ma Rui R Xu Sheng S Zhao Yucheng Y Xia Bing B Wang Ren R

Frontiers in plant science 20160425

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, ...[more]

PMID: 27200013

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Project description:Hulless barley (Hordeum vulgare L. var. nudum. hook. f.) has been cultivated as a major crop in the Qinghai-Tibet plateau of China for thousands of years. Compared to other cereal crops, the Tibetan hulless barley has developed stronger endogenous resistances to survive in the severe environment of its habitat. To understand the unique resistant mechanisms of this plant, detailed genetic studies need to be performed. The quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used method in detecting gene expression. However, the selection of stable reference genes under limited experimental conditions was considered to be an essential step for obtaining accurate results in qRT-PCR. In this study, 10 candidate reference genes-ACT (Actin), E2 (Ubiquitin conjugating enzyme 2), TUB? (Alpha-tubulin), TUB?6 (Beta-tubulin 6), GAPDH (Glyceraldehyde 3-phosphate dehydrogenase), EF-1? (Elongation factor 1-alpha), SAMDC (S-adenosylmethionine decarboxylase), PKABA1 (Gene for protein kinase HvPKABA1), PGK (Phosphoglycerate kinase), and HSP90 (Heat shock protein 90)-were selected from the NCBI gene database of barley. Following qRT-PCR amplifications of all candidate reference genes in Tibetan hulless barley seedlings under various stressed conditions, the stabilities of these candidates were analyzed by three individual software packages including geNorm, NormFinder, and BestKeeper. The results demonstrated that TUB?6, E2, TUB?, and HSP90 were generally the most suitable sets under all tested conditions; similarly, TUB? and HSP90 showed peak stability under salt stress, TUB? and EF-1? were the most suitable reference genes under cold stress, and ACT and E2 were the most stable under drought stress. Finally, a known circadian gene CCA1 was used to verify the service ability of chosen reference genes. The results confirmed that all recommended reference genes by the three software were suitable for gene expression analysis under tested stress conditions by the qRT-PCR method.

Project description:Quantitative real-time reverse transcription-polymerase chain reaction (qRT-PCR) is the most commonly used and powerful method for gene expression analysis due to its high sensitivity, specificity, and high throughput, and the accuracy of this approach depends on the stability of reference genes used for normalization. Taihangia rupestris Yu and Li (Rosaceae), an andromonoecious plant, produces both bisexual flowers and unisexual male flowers within the same individual. Using qRT-PCR technique, investigation of the gene expression profiling in staminate and perfect flowers would improve our understanding of the molecular mechanism in regulation of flower formation and sex differentiation in andromonoecious T. rupestris. To accurate normalize the gene expression level in Taihangia flower, 16 candidate reference genes, including 10 traditional housekeeping genes, and 6 newly stable genes, were selected based on transcriptome sequence data and previous studies. The expressions of these genes were assessed by qRT-PCR analysis in 51 samples, including 30 staminate and perfect flower samples across developmental stages and 21 different floral tissue samples from mature flowers. By using geNorm, NormFinder, BestKeeper, and comprehensive RefFinder algorithms, ADF3 combined with UFD1 were identified as the optimal reference genes for staminate flowers, while the combination of HIS3/ADF3 was the most accurate reference genes for perfect floral samples. For floral tissues, HIS3, UFD1, and TMP50 were the most suitable reference genes. Furthermore, two target genes, TruPI, and TruFBP24, involved in floral organ identity were selected to validate the most and least stable reference genes in staminate flowers, perfect flowers, and different floral tissues, indicating that the use of inappropriate reference genes for normalization will lead to the adverse results. The reference genes identified in this study will improve the accuracy of qRT-PCR quantification of target gene expression in andromonoecious T. rupestris flowers, and will facilitate the functional genomics studies on flower development and sex differentiation in the future.

Dataset Information

Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Lycoris aurea.

Publications

Selection and Validation of Appropriate Reference Genes for Quantitative Real-Time PCR Analysis of Gene Expression in Lycoris aurea.

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