Selecting stable reference genes for reverse transcription quantitative real‐time polymerase chain reaction analysis is critically important for gene expression. Bradysia odoriphaga is the most serious pest of Chinese chives, but our knowledge of its genetics is limited. In the present study, five housekeeping genes (β‐Actin, α‐Tubulin, RPS7, GAPDH and 18S rRNA) were cloned from B. odoriphaga using the combined techniques of reverse transcription polymerase chain reaction with rapid amplification of cDNA ends. The five genes, together with RPS15, were quantified for transcription stability in B. odoriphaga under various experimental conditions. Their expression stability was evaluated using four different algorithms (a comparative ΔCt method, GeNorm, NormFinder and BestKeeper). Additionally, we used an online Web‐based tool (RefFinder) to assign an overall final rank to each candidate gene. These analyses identified 18S, RPS15 and RPS7 as the most stable reference genes across developmental stages. 18S, RPS7 and Tubulin were the most stable reference genes for monitoring gene expression across different tissues of adults. RPS7, GAPDH and Tubulin were selected as the best reference genes across different larval tissues. GAPDH and RPS15 were selected to normalize gene expression for experiments of insecticide treatments. Actin and GAPDH are considered suitable reference genes in experiments of temperature treatments. Actin and Tubulin are considered suitable reference genes for starvation experiments. This study provides an important supplement of suitable reference genes for B. odoriphaga and these results provide clues toward the understanding of the genes’ biological functions in B. odoriphaga.

中文翻译：

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选择用于定量实时聚合酶链反应标准化的参考基因在 Bradysia odoriphaga（双翅目：Sciridae）中

选择稳定的参考基因进行逆转录定量实时聚合酶链反应分析对于基因表达至关重要。Bradysia odoriphaga 是韭菜中最严重的害虫，但我们对其遗传学的了解有限。在本研究中，使用逆转录聚合酶链反应与 cDNA 末端快速扩增的组合技术，从 B. odoriphaga 中克隆了五种管家基因（β-肌动蛋白、α-微管蛋白、RPS7、GAPDH 和 18S rRNA）。在各种实验条件下，这五个基因与 RPS15 一起被量化在 B. odoriphaga 中的转录稳定性。使用四种不同的算法（比较 ΔCt 方法、GeNorm、NormFinder 和 BestKeeper）评估它们的表达稳定性。此外，我们使用基于网络的在线工具 (RefFinder) 为每个候选基因分配一个总体最终排名。这些分析确定 18S、RPS15 和 RPS7 是整个发育阶段最稳定的参考基因。18S、RPS7 和微管蛋白是监测成人不同组织基因表达的最稳定的参考基因。RPS7、GAPDH 和 Tubulin 被选为不同幼虫组织的最佳参考基因。选择 GAPDH 和 RPS15 来标准化杀虫剂处理实验的基因表达。肌动蛋白和 GAPDH 被认为是温度处理实验中合适的参考基因。肌动蛋白和微管蛋白被认为是饥饿实验的合适参考基因。本研究为 B.