Total RNA was extracted from the harvested PG samples (n = 40) at different time points in B. mori development using Trizol reagent (Invitrogen, Carlsbad, CA) following the manufacturer's instructions. RNA concentration was further determined by spectrophotometer. Prior to first-strand cDNA synthesis, the purified RNA was treated with DNase for excluding genomic DNA contamination. First-strand cDNA synthesized from the total RNA (1 μg) of each PG sample using the PrimeScript RT reagent kit with gDNA Eraser (TaKaRa) was used as the template for qPCR. The primers for qPCR analysis are listed in Table S2. Since the ribosomal protein 49 (rp49) gene is the most stable in B. mori and has been widely used as a reference gene27, thus rp49 was chosen as a reference gene for normalization in our experiments. The efficiencies (E) of corresponding primers used in qPCR were calculated according to the equation: E = (10[−1/slope]−1) × 10028. qPCR was carried out using SYBR Green Supermix (TaKaRa) on an Applied Biosystems 7500 Fast Real-time PCR system (ABI, Carlsbad, CA, USA) according to the manufacturer's instructions. The thermal cycle conditions for qPCR were 95°C for 4 min, followed by 40 cycles of 95°C for 15 s and 60°C for 20 s. The specificity of the SYBR green PCR signal was further confirmed using agarose gel electrophoresis and melting curve analysis. The mRNA expression was quantified using the comparative Cross Threshold method (CT, the PCR cycle number that crosses the signal threshold)29. The CT of the rp49 gene was subtracted from the CT of the target gene to obtain ΔCT. The normalized fold changes of the target gene mRNA expression were expressed as 2−ΔΔCT, where ΔΔCT is equal to ΔCT treated sample −ΔCT control.