Selection and Validation of Suitable Reference Genes for RT-qPCR Analysis in the Rare Aquatic Firefly (Coleoptera: Lampyridae).

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 , , , , , , and for their expression stabilities in 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 and as the most appropriate reference genes for molecular studies in different life stages; and for different tissues; and for RNAi studies. The most appropriate reference genes in all treatments are and The results of this study will help improve accuracy and reliability to normalize RT-qPCR data in for further molecular analysis.