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Identification of appropriate reference genes for RT-qPCR analysis in Juglans regia L.

ABSTRACT: Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular adopted technique to detect gene expression, and the selection of appropriate reference genes is crucial for data normalization. In the present study, seven candidate reference genes were screened to evaluate their expression stability in various flower buds, leaf buds, tissues and cultivars of the English walnut (Juglans regia L.) based on four algorithms (geNorm, Normfinder, Bestkeeper and RefFinder). The results demonstrated that TUA, EF1 and TUB were appropriate reference genes for flower buds at different stages of female flower buds differentiation; TUB and 18S rRNA were best for leaf buds at different stages of female flower buds differentiation; TUB and TUA were suitable for different cultivars; and ACT2, 18S rRNA and GAPDH were useful for different tissues. Moreover, the expression of ACT was not stable among different flower buds, leaf buds and cultivars. The stability of reference genes were confirmed through the analysis of the expression of SPL18 gene. These results will contribute to a reliable normalization of gene expression in J. regia.

SUBMITTER: Zhou L

PROVIDER: S-EPMC6298729 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Identification of appropriate reference genes for RT-qPCR analysis in Juglans regia L.

Zhou Li L Niu Jianxin J Quan Shaowen S

PloS one 20181218 12

Reverse transcription quantitative real-time PCR (RT-qPCR) is a popular adopted technique to detect gene expression, and the selection of appropriate reference genes is crucial for data normalization. In the present study, seven candidate reference genes were screened to evaluate their expression stability in various flower buds, leaf buds, tissues and cultivars of the English walnut (Juglans regia L.) based on four algorithms (geNorm, Normfinder, Bestkeeper and RefFinder). The results demonstra ...[more]

PMID: 30562379

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Project description:BackgroundNitraria sibirica Pall. is a halophytic shrub with strong environmental adaptability that can survive in extremely saline-alkali and drought-impacted environments. Gene expression analysis aids in the exploration of the molecular mechanisms of plant responses to abiotic stresses. RT-qPCR is the most common technique for studying gene expression. Stable reference genes are a prerequisite for obtaining accurate target gene expression results in RT-qPCR analysis.ResultsIn this study, a total of 10 candidate reference genes were selected from the transcriptome of N. sibirica, and their expression stability in leaves and roots under different treatment conditions (salt, alkali, drought, cold, heat and ABA) was evaluated with the geNorm, NormFinder, BestKeeper, comparative ΔCt and RefFinder programs. The results showed that the expression stability of the candidate reference genes was dependent on the tissue and experimental conditions tested. ACT7 combined with R3H, GAPDH, TUB or His were the most stable reference genes in the salt- or alkali-treated leaves, salt-treated roots and drought-treated roots, respectively; R3H and GAPDH were the most suitable combination for drought-treated leaves, heat-treated root samples and ABA-treated leaves; DIM1 and His maintained stable expression in roots under alkali stress; and TUB combined with R3H was stable in ABA-treated roots. TBCB and GAPDH exhibited stable expression in heat-treated leaves; TBCB, R3H, and ERF3A were stable in cold-treated leaves; and the three most stable reference genes for cold-treated roots were TBCB, ACT11 and DIM1. The reliability of the selected reference genes was further confirmed by evaluating the expression patterns of the NsP5CS gene under the six treatment conditions.ConclusionThis study provides a theoretical reference for N. sibirica gene expression standardization and quantification under various abiotic stress conditions and will help to reveal the molecular mechanisms that confer stress tolerance to N. sibirica.

Dataset Information

Identification of appropriate reference genes for RT-qPCR analysis in Juglans regia L.

Publications

Identification of appropriate reference genes for RT-qPCR analysis in Juglans regia L.

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