不同实验条件下草莓(Fragaria × ananassa) qRT-PCR归一化合适内参基因的评价

IF 2.946 Q3 Biochemistry, Genetics and Molecular Biology BMC Molecular Biology Pub Date : 2018-06-22 DOI:10.1186/s12867-018-0109-4
Yunting Zhang, Xiaorui Peng, Yi Liu, Yali Li, Ya Luo, Xiaorong Wang, Haoru Tang
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引用次数: 38

摘要

草莓因其营养价值高、风味独特、外观美观而备受关注。全基因组序列和多个转录组数据库的可用性为全面探索基因功能提供了很大的可能性。靶基因的基因表达谱可以为理解其生物学功能提供线索。定量实时PCR (qRT-PCR)是快速定量基因表达的首选方法。该方法所获得结果的准确性要求具有稳定表达的内参基因对其数据进行规范化处理。本研究采用geNorm、NormFinder和BestKeeper三种统计算法,对7个候选内参基因在不同组织、果实发育阶段和不同低温处理植物中的表达稳定性进行了评价。我们的数据表明,在不同的实验条件下,内参基因的表达稳定性有所不同。总体而言,DBP、HISTH4、ACTIN1和GAPDH的表达更加稳定。在给定的实验条件下,由于稳定性低,不建议使用PIRUV、ACTIN2和18S进行归一化。此外,通过对HY5 (ELONGATED HYPOCOTYL5)的相对表达谱进一步证实了内参基因的可靠性,说明在qRT-PCR分析中,正确选用内参基因是非常重要的。草莓内参基因在不同条件下的表达稳定性不同。为了提高qRT-PCR分析的准确性和一致性,在计算靶基因表达水平之前,应对内参基因进行系统验证。
本文章由计算机程序翻译,如有差异,请以英文原文为准。

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Evaluation of suitable reference genes for qRT-PCR normalization in strawberry (Fragaria × ananassa) under different experimental conditions

Strawberry has received much attention due to its nutritional value, unique flavor, and attractive appearance. The availability of the whole genome sequence and multiple transcriptome databases allows the great possibility to explore gene functions, comprehensively. Gene expression profiles of a target gene can provide clues towards the understanding of its biological function. Quantitative real-time PCR (qRT-PCR) is a preferred method for rapid quantification of gene expression. The accuracy of the results obtained by this method requires the reference genes with consistently stable expression to normalize its data.

In present study, the expression stability of seven candidate reference genes in diverse sample subsets of different tissues and fruit developmental stages, and plant subjected to light quality and low temperature treatments was evaluated using three statistical algorithms, geNorm, NormFinder, and BestKeeper. Our data indicated that the expression stability of reference genes varied under different experimental conditions. Overall, DBP, HISTH4, ACTIN1 and GAPDH expressed much more stably. PIRUV, ACTIN2 and 18S were not recommended for normalization in given experimental conditions due to low stability. In addition, the relative expression pattern of HY5 (ELONGATED HYPOCOTYL5) was conducted to further confirm the reliability of the reference genes, which demonstrated the correct adoption of reference genes was of great importance in qRT-PCR analysis.

Expression stability of reference genes from strawberry varied across selected experimental conditions. Systematic validation of reference genes prior to calculation of target gene expression level should be done to improve the accuracy and consistency of qRT-PCR analysis.

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来源期刊
BMC Molecular Biology
BMC Molecular Biology 生物-生化与分子生物学
CiteScore
4.80
自引率
0.00%
发文量
0
审稿时长
>12 weeks
期刊介绍: BMC Molecular Biology is an open access journal publishing original peer-reviewed research articles in all aspects of DNA and RNA in a cellular context, encompassing investigations of chromatin, replication, recombination, mutation, repair, transcription, translation and RNA processing and function.
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