Screening and Identification of Reference Genes for Paracarophenax alternatus

In this study, we focused on the screening and identification of reference genes for <i>Paracarophenax alternatus</i> Xu and Zhang. The laboratory population was used as the laboratory population, and samples were collected from mites at four different stages, including physogastry, viviparous, 5 d viviparous and phoresy. Then, the expression levels of seven candidate reference genes (<i>α-tubulin</i>, <i>β-tubulin</i>, <i>RPS18</i>, <i>RPL13</i>, <i>GAPDH</i>, <i>EF1A</i>, <i>SDHA</i>) were detected through qRT-PCR. Melting curves showed good gene specificity, and the amplification efficiency ranged from 90% to 102%. ΔCt analysis indicated that <i>GAPDH</i> was the most stable reference gene. The GeNorm software determined that the optimal number of reference genes was two, with <i>GAPDH</i> and <i>RPS18</i> forming the most stable combination, and NormFinder identified <i>RPS18</i> as the most stable reference gene. Although the BestKeeper software suggested that <i>EF1A</i> was the most stable, its <i>p</i>-value exceeded 0.05, rendering it unsuitable for use as a reference gene. Finally, through the RefFinder network tool, the most stable reference genes were identified as <i>GAPDH</i> and <i>RPS18</i>.