牛奶子实时定量PCR分析中内参基因的评价与验证

doi:10.11707/j.1001-7488.20150516

Abstract

Abstract:

【Objective】 The accuracy of quantitative real-time polymerase chain reaction (qRT-PCR) analysis strongly depends on transcript normalization using stably expressed reference genes. The present study was conducted to evaluate the stability of candidate housekeeping genes and identify the most reliable gene or a set of genes to be used as reference genes in qPCR analysis of Elaeagnus umbellata.【Method】Twelve potential reference genes were selected among the most common reference genes reported in literature and their fragments were cloned by degenerate primers from E. umbellata, including 14-3-3, 18S ribosomal RNA gene (18 SrRNA), β-actin (Actin), elongation factor 1-α (EF 1-α), eukaryotic initiation factor 4A (eIF 4 A), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), RNA polymerase-Ⅱ (RP Ⅱ), 60S ribosomal protein L7 (RPL 7), translation elongation factor 2 (TEF 2), ubiquitin-conjugating enzyme E2 (UBCE), ubiquitin (UBQ) and ubiquitin extension protein 5 (UBQ 5). Samples were collected from five types of organs (root, stem, leaf, flower and red fruit), fruits at four different ripening stages (green, yellow, dark yellow and fully matured red fruit), green fruits at four time points after hormone ABA or GA₃treatments, detached leaves at four time points under 40 ℃, and three types of organs (root, stem and leaf) of the seedlings treated with salt stress at three time points. The expression stability of these 12 genes was evaluated based on the C_Tvalues using four statistical algorithms including geNorm, Normfinder, BestKeeper, and the comparative Δ C_T. Overall ranking of four sets aforementioned was generated using RefFinder software.【Result】 UBCE and RPL 7 were ranked as the two best reference genes for organ set, eIF 4 A and UBCE for fruit-ripening samples, eIF 4 A and UBCE for hormone treatment set, and Actin and EF 1-α for abiotic stress set. When including the data obtained from all the 29 samples into the analysis, eIF 4 A, RPL 7, and UBCE were identified as the top three reference candidates. The expression levels of phytoene synthase (EutPsy) were further assessed during fruit ripening by using the top three reference genes in comparison to the worst one RP Ⅱ. When the three stable reference genes eIF 4 A, UBCE and RPL 7 or the combination of top two eIF 4 A and UBCE were used for normalization, the trend of the relative transcript abundance of EutPsy were consistent. However, when the worst reference gene RP Ⅱ was employed for normalization, the expression profile was different. 【Conclusion】 eIF 4 A followed by RPL 7 and UBCE were found to be more reliable than other nine genes for normalization purposes in measuring gene expression of E. umbellata. This was the first systematic analysis for the selection of superior reference genes for qRT-PCR in E. umbellata under different conditions, which benefits future studies on gene expression in E. umbellata and other species of the genus Elaeagnus.

Key words: Elaeagnus umbellata, gene expression, qRT-PCR, normalization, reference gene

CLC Number:

S718.46

Cheng Longping, Hu Haitao, Guo Weidong, Yang Li, Wang Changchun, Yang Ling. Evaluation and Validation of Potential Reference Genes for Quantitative Real-Time PCR Analysis in Elaeagnus umbellata[J]. Scientia Silvae Sinicae, 2015, 51(5): 135-144.

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Evaluation and Validation of Potential Reference Genes for Quantitative Real-Time PCR Analysis in Elaeagnus umbellata

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