Hiscript 3 all in one rt supermix reagent kit

To optimize the ddPCR assay conditions, GAstV was used to collect leghorn male hepatocellular (LMH) cells infected with the GAstV XX strain. The infected LMH cells were extracted, the concentrations of RNA were quantified, and the viral nucleic acids were reverse transcribed. The rest of the collected virus and clinical samples were suspended in phosphate buffered saline (PBS, pH 7.4), and the virus or clinical samples were suspended in PBS at a ratio of 20%, w/v. The suspension was centrifuged at 4 °C at 8000 rpm for 15 min, after which the supernatant was retained. Viral nucleic acid and genomic DNA (FAdV-4, MDV-1, MDV-CVI988, and MDV-3/HVT) or RNA (GAstV, ALV, and AIV-H9N2) was extracted using the AxyPrep^TM Viral DNA/RNA Miniprep Kit (Axygen, Shanghai, China) according to the manufacturer’s instructions. Each viral nucleic acid RNA sample was reverse transcribed using the HiScript III All-in-one RT SuperMix reagent kit (Vazyme, Nanjing, China), following the product’s instructions. The cDNA/DNA was immediately amplified or stored at −80 °C for later use.

To validate the reliability of the transcriptome data, we chose 8 genes to measure their expression levels through qRT-PCR. Reverse transcription was performed with 1 μg of each RNA sample using the HiScript III All-in-one RT SuperMix reagent kit (Vazyme Biotech Co., Ltd., Nanjing, China). The target genes’ relative transcript levels were determined in triplicate using a 7500 Real-Time PCR equipment (ABI, Foster City, CA, USA). The actin gene (AB030227) served as an internal reference, and the data were analyzed using the 2^−ΔΔCT method.