Stepone plus thermal cycler

Total RNA was extracted from A549 with Trizol Reagent (Invitrogen) and real time RT-PCR was performed as described previously.^{9 (link),17 (link)} First-strand cDNA was synthesized from 2 μg of total RNA with Superscript III reverse transcriptase (Thermo Fisher Scientific) and oligo (dT)12–18. Real-time RT-PCR was carried out with cDNA synthesized from 50 ng of total RNA, SYBR Green PCR core reagents (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s protocol, and 0.2 μM specific primers for human ACE-2 (sense 5’-CATTGGAGCAAGTGTTGGATCTT-3’ and antisense 5’-GAGCTAATGCATGCCATTCTCA-3’) and β-actin (sense 5’-AGGCCAACCGCGAGAAGATGACC-3’ and antisense 5’-GAAGTCCAGGGCGACGTAGC-3’). The PCR thermal profile started with 10 min activation of Taq polymerase at 95°C followed by 40 cycles of denaturation at 94°C for 60 s, annealing at 55°C for 60 s, and extension at 72°C for 60 s, ending with dissociation curve analysis to validate the specificity of the PCR products. Reactions were performed in StepOne Plus Thermal Cycler (Thermo Fisher Scientific) and threshold cycle (Ct) data were collected. The relative ACE-2 expression was normalized to β-actin and calculated with the comparative Ct method of 2^−ΔΔCt.

Total RNA was extracted using RNeasy mini kit (Qiagen) as per the manufacturer’s instructions. RNA concentration was determined using Nanodrop One (Thermo Scientific). cDNAs were synthesized using iScript reverse transcription supermix (Bio-Rad) according to the manufacturer’s protocol. qRT-PCR was performed on StepOnePlus thermal cycler (Thermo scientific) with SYBR Select Master Mix (Thermo scientific). GAPDH was used as an internal control. Primer sequences are shown in Supplementary Table 2.

All qPCR was carried on the StepOnePlus thermal cycler (Life Technologies) with a PerfeCta SYBR Green FastMix kit (Quanta). Each reaction (total volume: 20 μl) comprised 1-fold SYBR Green qPCR mix, 300 nM both forward and reverse primer targeting toxR ( pirB-like, or ORF14-coding gene), and 1 μl of DNA template. The primer sequences are listed in Table 1. The following cycling conditions were employed: initial denaturation at 95°C for 3 min, followed by 40 cycles of 95°C for 5 s and 60°C for 30 s. For each gene, a standard curve of 6 serial dilutions of extracted DNA (from V. parahaemolyticus 13-028/A3) was run to determine the amplification efficiency for each gene. For each run, a non-template control was included, and each sample was run in duplicate. After quantitative PCR (qPCR) runs, melting curve analyses were performed to verify the presence of the amplicons. The Δ cycle threshold (Ct) values between the pirB-like (or ORF14-coding gene) and toxR gene (single-copy reference standard) were calculated by StepOne vs.2.2.2 software using a comparative Ct method.