Cfx96 manager software

The real-time PCR reaction was performed using the manufacturer’s protocol (Bio-Rad, Hercules, CA, USA). A total of 10 µL of the cDNA of the sample (10 ng cDNA/reaction) plus 10 µL of Ssoadvanced^™ universal SYBR Green Supermix was added to each well of the PrimPCR™ array plate. A PCR control assay template was added to the appropriate PCR control well. The plates were then sealed, mixed on the belly dancer machine, and centrifuged for 1 min at 10,000 rpm. Following these steps, the plate was loaded into the real-time PCR machine. The thermal cycling process, including the initial hold step, was set at 95 °C for 2 min and denaturation at 95 °C for 5 s, followed by 40 cycles of 60 °C for 30 s (annealing/extension) and 75 °C for 5 s/step (melting curve) using the Bio-Rad^™ CFX96 Real-Time System (Bio-Rad, Hercules, CA, USA). Finally, the Bio-Rad^® Mouse Inflammation, NF-κB, PI3k-Akt, mTOR, and NO signaling PrimePCR™ array data were analyzed via Bio-Rad CFX96 Manager software, version 3.1 (Bio-Rad, Hercules, CA, USA), which calculates fold change/regulation using the ΔΔCT (threshold cycle) method using the 2^−ΔΔCT formula. The p-values were calculated based on a student’s t-test of the replicate 2^−ΔCT values for each gene in the control group and experimental groups. PrimePCR™ array results were normalized to GAPDH using the Bio-Rad CFX96 Manager software.

The Novaplex SARS-CoV-2 Variants VII Assay (Novaplex) (Seegene, Seoul, Republic of Korea) was carried out according to the manufacturer’s instructions using the CFX96 Real Time PCR Detection System and the BioRad CFX96 Manager software (Bio-Rad, Hercules, CA, USA). The results were analyzed via automated data interpretation using Seegene Viewer software. The Novaplex assay is based on MuDT™ technology, which detects multiple targets with individual Ct values in a single channel without melting curve analysis. By utilizing the change in fluorescence signals between two different temperatures of detection, MuDT™ provides the “real” Ct value of each pathogen even in co-infected cases. MuDT™ allows for the genotyping of four SNP targets in a single tube. The set of primers can detect three SARS-CoV-2 variants of the S gene showing ∆69/70, E484A, and N501Y (Figure 1). The RdRP gene was considered as an internal control. All the NPSs that were only positive for the RdRP gene were classified as positive, but undetermined. Samples showing ∆69/70 deletion (Δ69/70) and N501Y were considered positive for the Alpha variant. Samples with ∆69/70, E484A, and N501Y SNPs were assigned to the Omicron BA.1 variant, while E484A and N501Y SNPs were attributed to the Omicron BA.2 variant.

Six genes with different expression profiles obtained by Illumina RNA-seq were selected for validation by qPCR. Gene-specific primers were designed by Primer premier 5.0. The Actin gene was used as housekeeping gene. Three biological and technical repetitions were used for each sample. Quantitative real-time PCR (qRT-PCR) was run on BioRad T100 (Bio-Rad Laboratories, Inc.) using SYBR Green Supermix according to the manufacturer’s instructions. The amplification program was set as follows: 95℃ for 5 min followed by 40 cycles of 95℃ for 15 s and 60℃ for 30 s. All reactions were performed with three independent biological replicates, and the expression levels calculated for each sample were based on three technical replicates. Data were analyzed using the Bio-Rad CFX96 Manager software (Bio-Rad Laboratories, Inc.). All data from qRT-PCR amplification were calculated with 2^−△△CT method [48 (link)].