Selection and Validation of Suitable Reference Genes for RT-qPCR Analysis in the Rare Aquatic Firefly Aquatica leii (Coleoptera: Lampyridae).
Selection and Validation of Suitable Reference Genes for RT-qPCR Analysis in the Rare Aquatic Firefly Aquatica leii (Coleoptera: Lampyridae).
复制标题
稀有水生萤火虫 Aquatica leii（鞘翅目：萤科）RT-qPCR 分析合适内参基因的选择和验证
DOI：
10.3390/insects12040359
复制
发表时间：
2021-04-16
期刊：
影响因子：
3
通讯作者：
Meyer-Rochow VB
中科院分区：
文献类型：
--
作者：
Fu X;Meyer-Rochow VB
本文旨在确定中国特有的雷氏水萤（Aquatica leii）中哪些基因适合作为未来实时荧光定量聚合酶链反应（RT - qPCR）研究中的内参基因。这是萤火虫有效基因敲低的首个实例，也是雷氏水萤后续研究在方法学上的一项重要进展。本研究结果将有助于提高雷氏水萤RT - qPCR数据标准化的准确性和可靠性，以用于进一步的分子分析。由于萤光素酶在萤火虫成虫发光器官中通常含量非常丰富，这是一个令人鼓舞的结果，不仅对雷氏水萤的研究人员有意义，对计划在萤火虫或其他非模式昆虫中进行RNA干扰（RNAi）或RT - qPCR的其他研究人员也有意义。
雷氏水萤（Aquatica leii Fu and Ballantyne）是一种珍稀的水生萤火虫，为中国特有种。它被认为是研究性闪光通讯系统分子机制的良好材料。为了改进保护和行为研究策略，需要进行涉及基因表达分析的大规模遗传学研究，而逆转录定量聚合酶链反应（RT - qPCR）是最常用的方法。然而，关于任何一种萤火虫的合适内参基因的报道都非常少。在此，我们评估了8种广泛使用的内参基因，包括18S、肌动蛋白（Actin）、Reep5、鸟氨酸脱羧酶1（Odc1）、微管蛋白（Tub）、甘油醛 - 3 - 磷酸脱氢酶（Gapdh）、延伸因子1α（Ef1a）和S27Ae，在三种不同条件下（即生活阶段、组织和双链RNA注射）在雷氏水萤中的表达稳定性。基于RefFinder计算的基因稳定性排名（RefFinder整合了四种算法：geNorm、ΔCt法、NormFinder和BestKeeper），我们推荐S27Ae和Reep5作为不同生活阶段分子研究中最合适的内参基因；Ef1a和Odc1用于不同组织；Tub和Odc1用于RNAi研究。在所有处理中最合适的内参基因是S27Ae和Tub。本研究结果将有助于提高雷氏水萤RT - qPCR数据标准化的准确性和可靠性，以用于进一步的分子分析。
The purpose of this paper was to determine which genes in Aquatica leii, an endemic Chinese firefly, are suitable for use as reference genes in future RT-qPCR studies. This is the first example of an effective knockdown in fireflies and an important methodological advance for continuing research in A. leii. The results of this study will help improve accuracy and reliability to normalize RT-qPCR data in A. leii for further molecular analysis. Since luciferase is very abundant in the adult light organ of fireflies generally, this is an encouraging result and of interest not just for A. leii researchers but other researchers who plan to perform RNAi or RT-qPCR in fireflies, or other nonmodel insects, as well. Aquatica leii Fu and Ballantyne is a species of rare aquatic firefly and endemic in China. It is considered good material to study the molecular mechanism of sexual flash communication systems. To improve conservation and behavioral research strategies, large-scale genetic studies involving gene-expression analysis are required and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) is the most commonly used method. However, there have been very few reports on appropriate reference genes in any species of firefly. Here, we evaluated eight widely utilized reference genes including 18S, Actin, Reep5, Odc1, Tub, Gapdh, Ef1a and S27Ae for their expression stabilities in A. leii under three different conditions, i.e., life stage, tissue and dsRNA injection. Based on the gene stability ranking calculated by RefFinder, which integrates four algorithms (geNorm, delta Ct method, NormFinder, and BestKeeper), we recommend S27Ae and Reep5 as the most appropriate reference genes for molecular studies in different life stages; Ef1a and Odc1 for different tissues; Tub and Odc1 for RNAi studies. The most appropriate reference genes in all treatments are S27Ae and Tub. The results of this study will help improve accuracy and reliability to normalize RT-qPCR data in A. leii for further molecular analysis.
登录
查看更多内容
影响因子：
14.8
作者：
通讯作者：
--
影响因子：
--
作者：
Silver N;Best S;Jiang J;Thein SL
通讯作者：
Thein SL
对 Aquatica leii（鞘翅目：萤科）水生幼虫捕食作为肝吸虫中间宿主的淡水蜗牛的形态和行为适应的调查
影响因子：
4.2
作者：
Fu, Xinhua;Meyer-Rochow, V. Benno
通讯作者：
Meyer-Rochow, V. Benno
影响因子：
3.7
作者：
Owens ACS;Meyer-Rochow VB;Yang EC
通讯作者：
Yang EC
复活萤火虫的古老光芒。
影响因子：
13.6
作者：
Oba Y;Konishi K;Yano D;Shibata H;Kato D;Shirai T
通讯作者：
Shirai T