Rt pcr kit

To explore the effect of T2 on PRV infection, PRV (MOI = 0.01) was used to inoculate cells in a 6-well plate with or without 10 nM T2. Total DNA of the PRV-infected cells was extracted, and the virus copy number was determined by quantitative PCR (qPCR). The standard curve was as follows: log(virus copy number) = −3.2428 Ct + 12.084 (R² = 0.995). The viral DNA in the PRV-infected cells and in the supernatants was extracted using commercial DNA extraction kits following the manufacturer’s instructions. The number of PRV DNA copies in the PK15 cells was determined by an RT-PCR kit (Accurate Biology, Changsha, China), as described previously. In brief, DNA extraction was performed using a DNA Mini kit (Accurate Biology, Changsha, China) and the purified DNA was used as a template for SYBR Green RT-PCR amplification. A 125 bp fragment of the PRV gB gene was amplified using forward and reverse primers (the prime sequences targeting to different genes are shown in Table 1). SYBR Green RT-PCR was performed using the ABI Prism Step One Plus detection system (Applied Biosystems, Shanghai, China). A recombinant PRV19 plasmid vector containing a PRV genome insertion was used as the standard.

After RBMSCs were seeded on the scaffolds for 7 and 14 days, the scaffolds were removed from the medium and washed twice with PBS. Total RNA from each group was extracted using an RNA extraction kit (AG21017, Accurate Biology, China) and quantified by a spectrophotometer (NanoDrop 2000; Thermo Fisher Scientific, Waltham, MA, United States). The obtained RNA was used to synthesize complementary DNA (cDNA) via an RT–PCR Kit (AG11706, Accurate Biology, China). Finally, RT–PCR was performed using SYBR^® Premix Ex TaqTM II (RR820A, Takara, Japan) in a CFX96 Real–time PCR machine (Bio–Rad, Hercules, CA, United States). The relative gene expression was calculated using 2^−∆∆Ct. GAPDH was used as the housekeeping gene, and the genes examined were RUNX2, BMP2, CD31 and VEGF. The primer sequences are detailed in Table 1.

Total RNA was extracted from the normal and PTC tissues by using Trizol Reagent (Accurate Biology, China). cDNA synthesis was performed using Reverse Transcription Kit (Accurate Biology, China). Quantitative RT-PCR was then carried out with the RT-PCR Kit (Accurate Biology, China). The sequence of all the primers used have been described in Table S3.