Transcription Analysis of Silkworm Larvae Exposed to Baculovirus

Total RNA was extracted from whole larvae treated with 10⁶ OBs/larva using the Trizol reagent (Invitrogen), as per the manufacturer’s protocol. Whole larvae samples were separately collected from silkworms in the TG-A, TG-B, TG-C, and WT groups treated with 10⁶ OBs/larva (n = 6 per group) harvested for analysis every 12 h from 0 to 72 hpi. RNA was quantified by spectrophotometry and purity was evaluated by gel electrophoresis. First-strand cDNA was prepared with the Thermo RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA) following the manufacturer’s protocol. To quantify the relative transcription levels of targeted genes, gene-specific primer sets were designed and used for qPCR performed with 2× SYBR Green PCR Master Mix (Toyobo). The housekeeping gene Rp49 (GenBank accession number AB048205.1) [28 (link)] was used as an internal control to standardize the variance of the different templates, and analyzed using the 2^−△△CT method [29 (link)]. The mRNA measurements were quantitated in three independent biological replicates and three independent technical replicates. Primers are listed in Supplementary Table S1.