Iqtm sybr green supermix kit

To measure the viral titer in respiratory and gastrointestinal tracts, nasal washes and rectal swab samples collected from ferrets were suspended in cold phosphate-buffered saline (PBS) containing antibiotics (5% penicillin/streptomycin; Gibco). To measure the viral copy number, total RNA was extracted from the collected samples using RNeasy Mini® kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions. A cDNA synthesis kit (Omniscript Reverse Transcriptase; QIAGEN, Hilden, Germany) was used to synthesize single-strand cDNA from total viral RNA. To quantify viral RNA copy number, quantitative real-time RT-PCR (qRT-PCR) was performed for the partial E gene primer set: forward primer, SARS-CoV-2-E-forward, atgtactcattcgtttcggaagag; and reverse primer, SARS-CoV-2-E-reverse, ctagagttcctgatcttctggtctaa with the SYBR Green kit (iQTM SYBR Green supermix kit, Bio-Rad, Hercules, CA, USA). The number of viral RNA copies was calculated and compared to the number of copies of the standard control.

To determine the expression levels of selected genes in NILs, total RNA was extracted from the shoot apex (<3 mm) of NILs at the R1 stage using Ribospin^TM Plant (GeneAll, Seoul, Korea), and cDNA was synthesized using Bio-Rad iScript^TM cDNA Synthesis Kit (Hercules, CA, USA). Next, qRT-PCR was performed on a LightCycler^® 480 (Roche Diagnostics, Laval, QC, Canada) using Bio-Rad iQ^TM SYBR Green Supermix Kit. Primer sequences were designed using PRIMER3plus (available online: http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) [48 (link)]. Appropriate primer pairs that did not amplify orthologues of target genes were selected using a stand-alone version of electronic PCR [49 (link)] (Table S5). Each qRT-PCR reaction mixture (20 µL volume) contained 100 ng cDNA template, and 300 µM each of forward and reverse primer. Amplification was performed using the following conditions: initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 10 s, and annealing and extension at 60 °C for 1 min. Three biological samples of each NIL were analyzed in triplicate to increase statistical power. The ACTIN11 (ACT11) gene was used as a reference for data normalization. Normalized data were analyzed using the method of Livak and Schmittgen [50 (link)]. Statistical significance was analyzed using Fisher’s least significant difference (p < 0.05) in R.

RNA from cultured cells or human tissue samples was prepared with TransZol (Trans Gen Biotech), and cDNA was synthesized from 5 µg of RNA using TransScript First-Strand cDNA synthesis SuperMix (Trans Gen Biotech). Gene expression was determined by real-time PCR using an iQTM SYBR Green SuperMix Kit (Bio-Rad, CA, USA) with a CFX96TM Real-Time system (Bio-Rad). All data were normalized to ACTB expression. The primers used are listed below. IL-6: forward primer 5’-GACAGCCACTCACCTCTTCA-3′, reverse primer 5’-AGTGC CTCTTTGCTGCTTTC-3’. MMP9: forward primer 5′-TTGACAGCGACAAGAAGTGG-3′, reverse primer 5′-GCCATTCACGTCGTCCTTAT-3′. SDHB: forward primer 5‘-ACAGCTCCC CGTATCAAGAAA-3’, and reverse primer 5‘-GCATGATCTTCGGAAGGTCAA-3’. ACTB: forward primer 5‘-TCCCTGGAGAAGAGCTACG-3’, and reverse primer 5‘-GTAGTTTCGTGG ATGCCACA-3’.

Total RNA was isolated using the PureZOL^TM RNA Isolation Reagent (Bio-Rad Laboratories, Inc. Hercules, Hercules, CA, USA) according to the manufacturer’s instructions and quantified through spectrophotometry (NanoDrop 2000, Thermo Scientific, Waltham, MA, USA). cDNA was synthesized using the iScript^TM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the supplier’s instructions, and was amplified using iQ^TM SYBR Green Supermix Kit (Bio-Rad) on iQ Thermal Cycler (Bio-Rad), according to the following program: initial denaturing step at 95.0 °C for 3 min; 40 cycles at 94.0 °C for 20 s; 54.0 °C for 30 s and 72.0 °C for 30 s. The primers, used at a final concentration of 10 μM, were: PLK1: forward 5′-CCCCTCACAGTCCTCAATAA-3′ and reverse 5′-TGTCCGAATAGTCCACCC-3′; GAPDH: forward 5′-ACAGTCAGCCGCATCTTC-3′ and reverse 5′- GCCCAATACGACCAAATCC-3′; Actin: forward 5′-AATCTGGCACCACACCTTCTA-3′ and reverse 5′-ATAGCACAGCCTGGATAGCAA-3′. Experiments were performed in triplicate, and the data were acquired using CFX ManagerTM Software (version 1.0, Bio-Rad). The results were analyzed according to ΔCT and normalized against Actin and GAPDH expression levels, which were used as control templates. A fold value of mRNA level ≥ or ≤1.5 relative to that of normal cells was considered as over- or underexpression, respectively.

To explore whether ARV infection and σA transfection affect the c-myc, HIF-1α, and glut1 of cancer cell lines (A549, B16-F10, and HeLa), cells were either infected with ARV at an MOI of 10 or transfected with the pCI-neo-σA plasmid. Cell lysates were collected at 24 h post infection. Total RNA was isolated from virus-infected or transfected cells using TRIzol reagent and subjected to quantitative real time RT-PCR with iQ^TM SYBR^® Green Supermix kit (Bio-Rad, Hercules, FL, USA) as described previously [16 (link)]. The specific primer pairs used in this work are shown in Table 1.

Kidneys were preserved at the time of collection in RNAlater, snap‐frozen in liquid nitrogen, and stored at 80°C. Total RNA was isolated from the kidneys with TRIzol reagent and treated with DNase to prevent genomic DNA contamination. cDNA was generated by reverse transcription of 1.5 µg RNA using M‐MLV reverse transcriptase. qPCR was performed using the Bio‐Rad iQTM SYBR^® Green Supermix kit according to the manufacturer's instructions. Gene expression was quantified using the Livak method (Livak & Schmittgen, 2001).
Following primers were used:

TRPV5	F:5′ CTGGAGCTTGTGGTTTCCTC 3′
	R:5′ TCCACTTCAGGCTCACCAG 3′
TRPM6	F:5′ CTTACGGGTTGAACACCACCA 3′
	R:5′ TTGCAGAACCACAGAGCCTCTA 3′
NCC	F:5′ CTTCGGCCACTGGCATTCTG 3′
	R:5′ GATGGCAAGGTAGGAGATGG 3′
CLDN16	F:5′ GTTGCAGGGACCACATTAC 3′
	R:5′ GAGGAGCGTTCGACGTAAAC 3′
CLDN19	F:5′ GGTTCCTTTCTCTGCTGCAC 3′
	R:5′ CGGGCAACTTAACAACAGG 3′
NCX1.3	F:5′ CTCCCTTGTGCTTGAGGAAC 3′
	R:5′ CAGTGGCTGCTTGTCATCAT 3′
Calbindin‐D28K	F:5′ GACGGAAGTGGTTACCTGGA 3′
	R:5′ ATTTCCGGTGATAGCTCCAA 3′
GAPDH	F:5′ TAACATCAAATGGGGTGAGG 3′
	R:5′ GGTTCACACCCATCACAAAC 3′