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用于蓟马(缨翅目:蓟马科)基因表达分析qRT-PCR标准化的内参基因的鉴定与验证

Identification and validation of reference genes for normalization of gene expression analysis using qRT-PCR in (thysanoptera: thripidae).

作者信息

Hou Qingfang, Yuan Linlin, Jin Haifeng, Yan Han, Li Fen, Wu Shaoying

机构信息

Sanya Nanfan Research Institute, Hainan University, Sanya, China.

School of Plant Protection, Hainan University, Haikou, China.

出版信息

Front Physiol. 2023 Apr 18;14:1161680. doi: 10.3389/fphys.2023.1161680. eCollection 2023.

Abstract

Gene expression analysis by reverse transcription quantitative polymerase chain reaction (qRT-PCR) has been widely used in research including insects. The selection of appropriate reference genes is the key to obtaining accurate and reliable results from qRT-PCR. However, studies on the expression stability of reference genes in remain lacking. In this study, qRT-PCR was used to analyze the expression stability of candidate reference genes in . The expression levels of six candidate reference gene transcription of were analyzed. GeNorm, NormFinder, BestKeeper, and ΔCt were used to analyze the expression stability of treated with biological factors (developmental period treatment) and abiotic factors (light, temperature, insecticide treatment, respectively). Comprehensive stability ranking of candidate reference genes was recommended by RefFinder. Results showed that ribosomal protein S () was the most suitable expression in insecticide treatment. Ribosomal protein L () was the most suitable expression at developmental stage and light treatment, whereas elongation factor was the most suitable expression in temperature treatment. RefFinder was used to comprehensively analyze the above four treatments, and the results showed that and actin () showed high stability in each treatment. Therefore, this study identified these two genes as reference genes in the qRT-PCR analysis of different treatment conditions of . Ourfindings will be beneficial for improving the accuracy of qRT-PCR analysis for future functional analysis of the target gene expression in .

摘要

通过逆转录定量聚合酶链反应(qRT-PCR)进行基因表达分析已在包括昆虫研究在内的众多研究中广泛应用。选择合适的内参基因是从qRT-PCR获得准确可靠结果的关键。然而,关于内参基因在[具体昆虫种类未提及]中的表达稳定性研究仍然缺乏。在本研究中,采用qRT-PCR分析了[具体昆虫种类未提及]中候选内参基因的表达稳定性。分析了六个候选内参基因转录本的表达水平。使用GeNorm、NormFinder、BestKeeper和ΔCt分析了经生物因素(发育期处理)和非生物因素(光照、温度、杀虫剂处理)处理后的[具体昆虫种类未提及]的表达稳定性。RefFinder推荐了候选内参基因的综合稳定性排名。结果表明,核糖体蛋白S([具体基因名称未提及])在杀虫剂处理中是最适合的表达内参。核糖体蛋白L([具体基因名称未提及])在发育阶段和光照处理中是最适合的表达内参,而延伸因子在温度处理中是最适合的表达内参。使用RefFinder对上述四种处理进行综合分析,结果表明[具体基因名称未提及]和肌动蛋白([具体基因名称未提及])在每种处理中均表现出高稳定性。因此,本研究确定这两个基因作为[具体昆虫种类未提及]不同处理条件下qRT-PCR分析的内参基因。我们的研究结果将有助于提高未来[具体昆虫种类未提及]中目标基因表达功能分析的qRT-PCR分析准确性。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5c56/10151585/4ce87fc8d67d/fphys-14-1161680-g001.jpg

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