Quantification of B. mori Transcripts

Ten annotated unigenes and five B. mori CYPs were randomly selected to quantify by qRT-PCR and evaluate data level. RNAiso plus reagent (TaKaRa) was used to extract total RNAs from three biological replications, and Primer Premier 5.0 was used to perform the primer design. qRT-PCR primers are displayed in Additional file 7 and Additional file 8. The reverse transcription system was used to synthesize cDNA as described previously [64 (link)–66 (link)]. qRT-PCR was performed on a LightCycler® 480 real-time PCR system (Roche). The reaction system consisted of 2 μl of cDNA, 10 μl of SYBR Green qPCR master mix (TaKaRa), 0.5 μl of each of the primers, and sterile water made up to 20 μl. The ΔΔCt method was used to analyze the relative differences in transcript levels [67 (link)]. Ribosomal protein L32 (Rpl32) was used for internal control, and the experiment was performed on three biological replicates.