Sybr select master mix reagent

BmN-SWU1 cells were plated in 6-well plates at a density of 1 × 10⁶ cells per well. After 24 hours, cells were infected with BmNPV at an MOI of 1. At the indicated time points, cells were harvested and total RNA was prepared using TRIzol reagent (Invitrogen, USA) as described previously43 (link). Samples were normalized to total RNA and reverse transcription reactions were performed with SYBR Select Master Mix Reagent (Bio-Rad, USA) using specific primers (S1 Table). The Bombyx mori sw22934 gene was used as an endogenous control. The sample analysis was performed in triplicate on the CFX96 Real-Time System.

B. mori ribosomal protein L3 (BmRpL3) was used as an internal control for normalization. The 20 μL mixture included 2 μL cDNA, 0.5 μL each primer (10 mM; Table S3), 10 μL SYBR™ Select Master Mix reagent (Bio-Rad, Hercules, CA, USA) and 7 μL ddH₂O. RT-qPCR was performed according to the following parameters: one cycle of an initial denaturation step at 95 °C for 1 min, 40 cycles at 95 °C for 30 s and 60 °C for 20 s and a final cycle at 95 °C for 15 s, 60 °C for 30 s and 95 °C for 15 s. The specific primers of genes for qPCR are listed in Table S3. The relative gene expression levels were estimated according to the 2^−ΔΔCt method [45 (link)]. All samples were run in triplicate.

The qPCRs were conducted in a CFX96 real-time system using the SYBR select master mix reagent (Bio-Rad, Hercules, CA, USA) to determine N. bombycis genome copy numbers. Specifically, N. bombycis small subunit rRNA (SSUrRNA) gene-specific primers were used to amplify rRNA genes and estimate genome replication to ultimately evaluate genome copy numbers after 48 h of infection (41 (link)). A standard curve described by y = −3.4305× + 36.882 (R² = 0.9989) was used to evaluate the expression levels. The sw25113 gene was used as an internal reference to determine the relative gene expression from qPCRs data. qPCRs were conducted at 95°C for 30 s, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s.