Luna universal qpcr 2x master mix

Quantification of expressed genes was carried out in the CFX96™ Real-time PCR system and software (Bio-Rad, Hercules, CA, USA). RT-qPCR reactions were performed according to the manufacturer’s instructions using a Luna^® Universal SYBR green qPCR 2X Master mix (New England Biolabs, Ipswich, MA, USA) in a total volume of 20 μL, containing 10 μL of Luna Universal qPCR 2X Master Mix, 100 ng of cDNA template, 0.25 μM of forward primer, 0.25 μM of reverse primer and sterile nuclease-free water to complete 20 μL. cDNA amplification involved an incubation for initial denaturation at 95 °C for 1 min, followed by 40 cycles of 95 °C for 15 s, and 55–60 °C for 45 s. After 40 cycles, a melting curve was determined using SYBR green fluorescence. Negative controls for gene quantification were performed by omitting the cDNA template from an amplification reaction. Normalization of amplification curves of genes was determined using S. aureus (muc) and E. coli (rssA) housekeeping reference genes. Gene expression and quantification of amplification efficiency were carried out using the 2-ΔΔCt method [82 (link)].

Viral progeny was quantified by qPCR. Seventy-two hours post-transfection, cell culture supernatants were harvested and clarified by centrifugation at 3,000g for 10min. Supernatants were treated with lysis buffer (50mm Tris-HCl pH 7.5; 1mm EDTA; 1% SDS; 0.5mg/ml proteinase K (Invitrogen, United States)) and incubated at 56°C for 2h. Nucleic acids were purified by phenol-chloroform extraction and ethanol precipitation in the presence of 20μg of dextran. qPCR was performed in a Step One Plus Real-Time PCR System (Applied Biosystems, Foster City, CA, United States) using Luna Universal qPCR Master Mix (2x; New England Biolabs, Beverly, MA, United States). The following primers were used for the amplification: sense 5’-ATGGAGACCACCGTGAACGC-3′ (nt 1608–1627) and antisense 5′-AGGCACAGCTTG GTGGCTTG-3′ (nt 1887–1868). To avoid amplification of the input linear HBV DNA used for transfection, primers that specifically amplify relaxed circular HBV DNA were used (Elizalde et al., 2019 (link)). Serial dilutions of an HBV replication-competent plasmid (pCH-9/3091) were used as quantification standards.