Bioeasy sybr green 1 real time pcr kit

The in vitro viral challenge assay was strictly performed at a designated safe place. TG fibroblasts, TG kidney cells and TG umbilical vein endothelial cells were isolated from newborn TG pigs. These cells, cultured in 24-well plates, were inoculated with 200 TCID₅₀ of CSFV (Shimen strain), and there were four replicates for each TG cell types. One hour later, the inoculums were replaced with fresh medium (5% fetal bovine serum). After 48-h incubation, cells and virus were collected and evaluated by IFA and qPCR. To analyze CSFV proliferation in TG cells by qPCR, total RNA was extracted from the CSFV-infected cells using TRIzol-A+ reagent (Tiangen, Beijing, China) and reverse transcribed into cDNA using the BioRT cDNA First Strand Synthesis Kit (Bioer, Hangzhou, China) according to the manufacturer’s protocol. SYBR Green real-time PCR was performed using the BIO-RAD IQ5 multicolor real-time PCR detection system and the BioEasy SYBR Green I real-time PCR kit.

Quantitative RT-PCR was performed to examine CSFV in pig blood. Blood samples from each pig were collected at days 0, 2, 4, 6, 8, 10, 12, 14, 16 and 18 after injection. Viral genomic RNA was isolated by using Trizol (Tiangen, Beijing, China) according to the manufacturer’s instructions. A standard curve was generated to detect the viral load in each blood sample with 10-fold serial dilutions of viral lysates ranging from 10⁸ to 10². SYBR Green real-time PCR was performed using the BIO-RAD IQ5 multicolor real-time PCR detection system and the BioEasy SYBR Green I real-time PCR kit and the Ct values and CSFV RNA copies were determined.

Total RNA was extracted from cells using the AllPrep DNA/RNA Micro Kit (QIAGEN, Germany) according to the manufacturer’s instructions. cDNA was synthesized using the First-Strand cDNA Synthesis kit (Promega, U.S.A.). Quantitative real-time PCR (qRT-PCR) was performed to determine H19, miR675, and m⁶A-related gene expression using the BioEasy SYBR Green I Real-Time PCR Kit on Bio-Rad iQ5 Multicolor Real-Time PCR Detection System (Bioer Technology, China). The miR675 3p and 5p sequences are listed in Supplementary Table S2, and the miRNA primer sequences are listed in Supplementary Table S3. The primer sequences of m⁶A-related genes and H19 are listed in Supplementary Table S4. For PCR, the initial denaturation was conducted at 95°C for 3 min, followed by 40 cycles of denaturation at 95°C for 10 s, annealing at 60°C for 15 s, and extension at 72°C for 30 s. The 2^−ΔΔC_T method was used to determine relative gene expression. The experiments were performed at least in triplicates.