| Summary: | Zygentoma occupies a key evolutionary position for understanding the evolution of insect metamorphosis but has received little attention in terms of genetic analysis. To develop functional genomic studies in this insect, we evaluated five candidate internal reference genes for quantitative RT-PCR (qPCR) studies from <i>Thermobia domestica</i>, a representative species of Zygentoma, including <i>Actin 5C</i> (<i>Actin5C</i>), <i>Elongation factor-1 alpha</i> (<i>EF1A</i>), <i>Ribosome protein S26</i> (<i>RPS26</i>), <i>Ribosome protein L32</i> (<i>RPL32</i>), and <i>Superoxide dismutase 2</i> (<i>SOD2</i>), at different developmental stages, in various body parts, and under dsRNA microinjection and starvation stresses, using four algorithms (delta Ct, geNorm, NormFinder and BestKeeper) and a comparative algorithm (RefFinder). Specific suitable reference genes were recommended across specific experimental conditions, and the combination of <i>RPS26</i> and <i>RPL32</i> was appropriate for all tested samples. Employing our selected reference gene combination, we investigated the gene expression pattern of <i>Myoglianin</i> (<i>Myo</i>), a crucial gene-regulating insect metamorphosis, in ametabolous <i>T. domestica</i>, and demonstrated the efficiency of RNA interference (RNAi) in firebrat nymphs. This study provides a basis for reliable quantitative studies of genes and greatly benefits evolutionary and functional genomics studies in Zygentoma.
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