Cellular membrane cholesterol is required for porcine reproductive and respiratory syndrome virus entry and release in MARC-145 cells

Sun, Ying; Xiao, ShaoBo; Wang, Dang; Luo, Rui; Li, Bin; Chen, HuanChun; Fang, LiuRong

Total RNA was extracted from cell suspensions or supernatant with RNAprep Micro Kit (Tiangen, Beijing, China), according to the manufacturer's instructions. RNA was re-versetranscribed into cDNA using ReverTra Ace-α (Toyobo, Osaka, Japan) with Oligo (dT) 20 . Quantitative real-time PCR was performed according to protocols described by Egli et al. [33] . In brief, the generated cDNA was amplified by quantitative real-time PCR using primers for the PRRSV ORF7 gene, reverse primer (5′-AAATGGGGCTTCTCCG-GGTTTT-3′) and forward primer (5′-TCAGCTGTGCCA-AATGCTGG-3′), and specific fluorescent probe (5′-TCC-CGGTCCCTTGCCTCTGGA-3′; sense orientation), which was 5′-labeled with 6-carboxyfluorescein (FAM; reporter) and tetramethylrhodamine (TAMRA) at its 3′-end. Real-time PCR was performed in a single tube in an ABI Prism 7500 Real-Time PCR System (Applied Biosystems, Foster City, CA). For each run, four no-template controls containing water instead of DNA, as well as five PRRSV ORF7 gene standards corresponding to 10 12 , 10 11 , 10 10 , 10 9 , 10 8 copies per tube were included. Samples of interest were run in triplicate. Data from the TaqMan ® run were analyzed using the SDS software (Version 1.7; Applied Biosystems).