Transstart one step gdna removal and cdna synthesis supermix kit

Total RNA was isolated from MDBK cells using the Omega total RNA kit (R6834, Guangzhou, China). For the cDNA synthesis, 1 µg of total RNA was used for reverse transcription by using a TransStart one-step gDNA removal and cDNA synthesis supermix kit (AT311-02, TransGen Biotech, Beijing, China). Quantitative real-time RT-PCR was performed using the 10 µL SYBR Green PCR Master Mix (LS2062, Promega, Madison, WI, USA), 0.5 µL of forward or reverse primer, 1 µg cDNA, and an addition of H2O to a final reaction volume of 20 µL. This study used the following primers: Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (forward: 5′-AAAGTGGACATCGTCGCCAT-3′, reverse: 5′-CCGTTCTCTGCCTTGACTGT-3′), BVDV 5′UTR (forward: 5′-TAGTCGTCAGTGGTTCGACGCC-3′, reverse: 5′-CCTCTGCAGCACCCTATCAG-3′). IFITM3 (forward: 5′-ATCCCAGCCCTTGTTCACTG-3′, reverse: 5′-GACACGAGGACGATGAGGAC-3′). The results were analyzed by the cycle threshold (CT) method.

Total RNA was extracted from cells using the Omega total RNA kit, and cDNA was then synthesized using a TransStart one-step gDNA removal and cDNA synthesis supermix kit (TransGen Biotech) according to the manufacturer’s instruction. qRT-PCR was performed in triplicate using TransStart Tip Green qPCR supermix (TransGen Biotech) following the manufacturer’s protocols. The results were normalized against the level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) expression and quantified by the threshold cycle (2^−ΔΔCT) calculation method. The primers used for qRT-PCR were as follows: GAPDH-F (5′-ACATGGCCTCCAAGGAGTAAGA-3′) and GAPDH-R (5′-GATCGAGTTGGGGCTGTGACT-3′), PDCoV N-F (5′-CGCTTAACTCCGCCATCAA-3′) and PDCoV N-R (5′-TCTGGTGTAACGCAGCCAGTA-3′), ABCA1-F (5′-CTGCCTCCTCCACAGAGAAAAC-3′) and ABCA1-R (5′-CAGGGAAAACCCACCATACCT-3′), ACAT1-F (5′-CTGGGTGCAGGCTTACCTAT-3′) and ACAT1-R (5′-ACATGCTCTCCATTCCACCTG-3′), ACAT2-F (5′-AGCAGGTTGGTCACTGGAAG-3′) and ACAT2-R (5′-TCCTCCTTCAGTGTTGACCTTT-3′), SREBF2-F (5′-TTGTCGGGTGTCATGGGCG-3′) and SREBF2-R (5′-ATTGCAGCATCTCGTCGATGT-3′), and IFN-β-F (5′-TTCGAGGTCCCTGAGGAGATT-3′) and IFN-β-R (5′-TCCATCTGCCCATCAAGTTCC-3′).

The TransStart one-step gDNA removal and cDNA synthesis supermix kit (AT311-02) and TransStart Tip Green qPCR supermix (AQ142-21) were ordered from TransGen Biotech (Beijing, China), The total RNA kit was ordered from Omega (Guangzhou, China), BODIPY 493/503 (GC42959) was purchased from GlpBio (Montclair, CA, USA), DAPI (4′,6-diamidino-2-phenylindole) (C0060) was ordered from Solarbio Science & Technology (Beijing, China), and 25-hydroxycholesterol (25HC) (HY-113134) and asiaticoside (AS) (HY-N0439) were ordered from MedChemExpress (Monmouth Junction, NJ, USA).