Real-time quantitative PCR (qPCR) is a reliable and widely used technique for analyzing the expression profiles of target genes in different species, and reference genes with stable expressions have been introduced for the normalization of the data. Therefore, stability evaluation should be considered as the initial step for qPCR experiments. The fall armyworm Spodoptera frugiperda (J. E. Smith) (Lepidoptera: Noctuidae) is a polyphagous pest that consumes many plant species and seriously threatens corn production around the world. However, no studies thus far have examined the stability of reference genes in this pest. In this study, the expression profiles of the eight candidate reference genes of Actin, elongation factor 1 alpha (EF1α), elongation factor 2 (EF2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ribosomal protein L3 (RPL3), ribosomal protein L13 (RPL13), alpha-tubulin (α-TUB), and beta-1-tubulin (β-1-TUB) were obtained from S. frugiperda in different samples and the stability was evaluated by ΔCt, BestKeeper, geNorm, NormFinder, and RefFinder methods. The results of pairwise variation (V) calculated by GeNorm indicated two reference genes could be selected for normalization. Therefore, the combinations of the most stable reference genes for different experimental conditions of S. frugiperda were shown as follows: EF2 and RPL13 for developmental stages, RPL3 and β-1-TUB for larval tissue samples, EF2 and EF1α for the larval samples treated with different temperatures, RPL3 and EF1α for the larval samples under starvation stress, and RPL13 and EF1α for all the samples. Our results lay the foundation for the normalization of qPCR analyses in S. frugiperda and could help guarantee the accuracy of subsequent research.

实时定量 PCR (qPCR) 是一种可靠且广泛使用的技术，用于分析不同物种中靶基因的表达谱，并引入了具有稳定表达的参考基因来对数据进行标准化。因此，稳定性评估应被视为 qPCR 实验的初始步骤。秋季粘虫草地贪夜蛾（JE Smith）（鳞翅目：夜蛾科）是一种多食性害虫，消耗许多植物物种，严重威胁世界各地的玉米生产。然而，到目前为止，还没有研究检查过这种害虫中参考基因的稳定性。在本研究中，肌动蛋白、延伸因子 1 α ( EF1α )的 8 个候选参考基因的表达谱，延伸因子 2 ( EF2 )、3-磷酸​​甘油醛脱氢酶( GAPDH )、核糖体蛋白 L3 ( RPL3 )、核糖体蛋白 L13 ( RPL13 )、α-微管蛋白( α-TUB ) 和β-1-微管蛋白( β-1) -TUB ) 来自 S. frugiperda在不同样品中，稳定性通过 ΔCt、BestKeeper、geNorm、NormFinder 和 RefFinder 方法进行评估。GeNorm 计算的成对变异 (V) 结果表明可以选择两个参考基因进行标准化。因此，对于草地早熟禾不同实验条件的最稳定的参考基因组合如下：EF2和RPL13用于发育阶段，RPL3和β-1-TUB用于幼虫组织样品，EF2和EF1α用于处理的幼虫样品不同温度下，饥饿胁迫下幼虫样品的RPL3和EF1α，以及所有样品的RPL13和EF1α。我们的结果为草地贪夜蛾qPCR 分析的标准化奠定了基础，有助于保证后续研究的准确性。