Hiscript 3 all in one rt supermix perfect for rt qpcr

To validate the RNA-seq data, three miRNAs were randomly selected for RT-qPCR confirmation with ChamQ™ universal SYBR^® RT-qPCR master mix (Vazyme, China). Total RNAs were extracted using RNA Isolater Total RNA Extraction Reagent (Vazyme, China). For miRNA determination, 1 µg of total RNA was reversed using the miRNA 1st Strand cDNA Synthesis Kit (by stem-loop) (Vazyme, China) with miRNA-specific stem-loop primers. For gene quantification, the cDNA was prepared using HiScript^® III All-in-one RT SuperMix Perfect for RT-qPCR (Vazyme, China) with the gDNA wiper. The PCR was conducted with a StepOnePlus™ Real-Time PCR System (Applied Biosystems, Thermo Fisher Scientific, Waltham, MA, USA) under the following conditions: 95 °C for 30 s, followed by 40 cycles of 95 °C for 10 s and 60 °C for 30 s. The specificity of each primer pair was ensured by analyzing the melting curve. Experiments were performed with three independent biological replicates and technical replicates. The relative quantity of miRNAs and mRNAs was analyzed by RT-qPCR and β-actin was used as the reference gene. All primers were synthesized by the Sangon Biotech Co., Ltd. (Shanghai, China) (Table S1). Data were analyzed by the 2^−ΔΔCT method.

Conidia of WT and BbVRF1 strains were harvested, and the concentrations of their suspensions were adjusted to 2 × 10⁶ conidia mL⁻¹. For the in vitro experiment, H. armigera cells were cultured in six-well culture plates (60,000 cells per well) and infected with 1 μL suspension of WT or BbVRF1. The mock-infected (infected with 1 × phosphate-buffered saline [PBS]), WT-infected (infected with WT strain), and BbVRF1-infected (infected with BbVRF1 strain) cells were collected 72 h later. For the in vivo experiment, 5th-instar H. armigera larvae were injected with 5 μL conidia suspension (2 × 10⁶ conidia mL⁻¹) of WT and BbVRF1. The hemocytes were collected at 0 h, 12 h, 24 h, 48 h and 72 h after infection. RNA was extracted from samples using the RNA Easy Fast tissue/cell kit (Tiangen, China). The RNA was treated using the HiScript III All-in-One RT SuperMix Perfect for RT-qPCR (Vazyme, China) for reverse transcription, and qRT-PCR was performed using the StepOne Plus RT-qPCR system (Applied Biosystems, USA) and Taq Pro Universal SYBR qPCR master mix (Vazyme, China) according to the manufacturers’ manuals, using the primers listed in Table S1. The rps3 gene was used as an internal reference (35 (link)).

Viral nucleic acids were extracted from cultured viruses; clinical samples oral, nasal, and anal swabs; tissue specimens; environmental swabs; and serum samples—using the DNA/RNA Extraction Kit (Prepackaged) (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China) following the manufacturer’s instructions. The extracted viral nucleic acids were eluted in 100 μL of nuclease-free double-distilled water (ddH₂O) and stored at −80 °C until further use. The synthesis of cDNA was carried out using HiScript^® III All-in-one RT SuperMix Perfect for RT-qPCR (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China), with a reverse transcription program including reaction incubation at 50 °C for 15 min, followed by 85 °C for 5 s.