Cfx96 qpcr machine

NT swabs from all suspected COVID-19 patients underwent rRT-PCR testing for SARS-CoV-2 at the TRC-EID-HSC or at the microbiology laboratory of the Faculty of Medicine, Chulalongkorn University. At TRC-EID-HSC Nucleic acid (NA) extraction was performed on all samples using magLEAD kit according to the manufacturer’s instructions (Precision System Science) and the RT-qPCR was performed using Seegene—Allplex 2019-nCov Assay, in a Bio-Rad CFX 96 qPCR machine according to manufacturer’s instruction. Briefly, reaction was heated to 50oC for 20 minutes for reverse transcription, denatured in 95oC for 15 minutes and then 45 cycles of amplification was carried in 94oC for 15 seconds and 58oC for 30 seconds. Fluorescence was measured using four fluorescence channels: FAM (E gene), HEX (internal control), Cal Red 610 (RdRp gene), Quasar 670 (N gene). The protocol’s stated limit of detection of ORF1ab rRT-PCR was 1000 copies/mL and the cut-off PCR cycle threshold (Ct) was 40.
At the microbiology laboratory the cobas® 6800 system (Roche Diagnostics, Switzerland) was used for detection of SARS-CoV-2 RNA which targeting conserved regions within the ORF 1a/b and E genes, according to manufacturer’s instruction.

RNA was extracted using Trizol (Invitrogen) following the manufacturer's instructions. One microgram of each mRNA sample was used for synthesis of first-strand cDNA using the MultiScribeRT enzyme (Applied Biosystems). qPCR amplification (40 cycles of 15 sec at 95°C and 1 min at 60°C) was performed using the Bioline SensiMix II reagent (Celtic Diagnostics) in a CFX96 qPCR machine (BioRad). The housekeeping gene cyclophilin A (CYPA) was used for normalization. The names of genes, their accession number and primers used are indicated in Table 1. The TaqMan array for Human Extracellular Matrix & Adhesion Molecules (4414133, Life Technologies) was performed using the same qPCR machine and conditions described above.

qPCR was performed using a previously described duplex assay^{25 (link)} with the primers and probes given in Supplementary Table S3. VLPs were lysed by heating to 95°C for 5 min in a thermocycler before being added to qPCRs. qPCRs (20 μL) used TaqPath™ qPCR Master Mix, CG (ThermoFisher Scientific) with final primer and probe concentrations of 400 and 200 nM, and a sample volume of 1 μL. The reaction mixture was then thermocycled (50°C for 2:00, 95°C for 5:00 and 45 cycles of 95°C for 0:10, and 60°C for 1:00) on a BioRad CFX96 qPCR machine.

Total RNA was isolated using TRIzol^TM Reagent (Invitrogen, Thermo Fisher Scientific) according to the manufacturer’s instructions. cDNA was synthesized using MMLV reverse transcriptase (Promega, Madison, WI, USA) after which qRT-PCR was performed in triplicate for CDH1, SNAI2, and GAPDH genes using an iQ SYBR Green Supermix and a CFX96 qPCR machine (BioRad Laboratories, Hercules, CA, USA). The used primers are as follows: forward, 5′-TCAGCGTTGTGACTGTGAA-3′ and reverse, 5′-CCTCCAAGAATCCCCAGAAT-3′ for CDH1; forward, 5′-TCTGCAGACCCATTCTGATG-3′ and reverse, 5′-AGCAGCCAGAT TCCTCATGT-3′ for SNAI2 and forward, 5′-ACAGTCAGCCGCATCTTCTT-3′ and reverse, 5ʹ-ACGACCAAATCCGT TGACTC-3′ for GAPDH. The following amplification conditions were used: an initial denaturation step at 95 °C for 3 min, followed by 40 cycles of denaturation at 95 °C for 10 s, annealing at 56 °C for 10 s, and extension at 72 °C for 10 s. The 2^-ΔΔCt method was used to calculate mRNA expression levels using GAPDH as the reference gene.