Cfx manager software v3

The qPCR Sybr Green assay was utilized to measure the 16S rDNA gene copy number in 25 μl of sample using the CFX96 Touch Real Time PCR Detection System (Bio-Rad, United States). Reactions contained 5 μl of DNA template (from 50 to 5 ng DNA for reaction tube), 12.5 μL of 2X SYBR Green Supermix (Bio-Rad, United States), and primers at required concentrations (Di Cesare et al., 2015 (link)). Triplicates samples and no template controls (NTCs) were analyzed. The E. coli 16S rDNA was used as positive control, and standard curves were produced with gene copy numbers from 10² to 10⁶ genes per reaction tube. The concentration of the amplified DNA was determined using NanoDrop spectrophotometer. The gene copy number per μl of solution was calculated according to literature reports (Czekalski et al., 2012 (link)). The qPCR results were reported as the mean of measurements of triplicates analysis with standard deviations. Data were analyzed with the CFX ManagerTM software v3.1 (Bio-Rad, Italy).

Thermal unfolding profiles of hPAH were obtained by DSF, in a C1000 Touch thermal cycler with a CFX96 optical reaction module (Bio-Rad; Hercules, CA, USA) as described in [10 (link)]. All assays were carried out in SEC buffer and with Sypro Orange at a 2.5× final concentration. The PCR plates were sealed with Optical-Quality Sealing Tape (Bio-Rad) and centrifuged at 500× g for 5 min. The thermal profiles were obtained by ramping the temperature between 20 and 90 °C at 1 °C/min, with a 1 s hold time every 0.2 °C and fluorescence acquisition through the FRET channel. Data were analysed with CFX Manager Software V3.0 (Bio-Rad) and GraphPad Prism software V6.00 (La Jolla, CA, USA), fitting the experimental curves with a biphasic dose–response function to obtain the hPAH T_ms from the midpoint of the first (T_m1) and second (T_m2) transitions.

Differential scanning fluorimetry (DSF) is a methodology whereby a fluorescent dye binds to the proteins buried hydrophobic residues that become exposed upon thermal unfolding. DSF assays were performed in a C1000 Touch thermal cycler equipped with a CFX96 optical reaction module (Bio-Rad, Hercules, CA), by having the GALT variants at a 0.1 mg/mL (∼2.5 μmol/L in monomer) final concentration in buffer A, SYPRO orange (Invitrogen Corporation, Carlsbad, CA) at a 5× working concentration (Niesen et al. 2007 (link)), in a 50 μL total volume. A 10-min incubation step at 20°C preceded the temperature ramp from 20 to 90°C at 1°C/min, with a 1-sec hold time every 0.2°C and fluorescence acquisition using the HEX channel (excitation maximum at 535 nm, emission maximum at 555 nm). Assays using 2.0 mmol/L Gal-1-P, 0.5 mmol/L UDP-Glc, 100 μmol/L Fe²⁺, and 100 μmol/L Zn²⁺ were also performed. Control assays in the absence of protein were routinely performed. Data were processed using CFX Manager software V3.0 (Bio-Rad). Temperature scan curves were fitted to a biphasic sigmoidal function and the T_m values were obtained from the inflexion points of the first and second transitions. Variations in T_m values are considered significant when |ΔT_m| ≥ 2°C (above the standard deviation).

A real-time PCR protocol was setup using D. suzukii positive control samples as well as several non-target species samples as negative controls. The designed primer pairs that amplify D. suzukii were used in a real-time PCR run on a CFX96 Touch Real-time platform (BioRad, Hercules, CA, USA). Optimisation gradients of temperature (55–65°C), primer concentration (50 nM–400 nM) and Mg²⁺ concentration (3 mM–5 mM) were run to optimise the PCR conditions using the SsoFast EvaGreen Supermix (BioRad). The assay was also preliminarily tested using the Accumelt HRM Supermix (Quanta Biosciences, Gaithersburg, MD, USA) which employs the SYTO9 green fluorescent dye and Platinum Quantitative PCR SYBR Supermix UDG (Invitrogen), which employs the SYBR Green I dye. The performance of the optimised assay on these mastermixes was compared to select the optimal mastermix.
For each reaction, 96-well clear bottom plates were used and all samples, standards and controls were run in duplicate wells. Cycling protocol including the HRM protocol was developed based on the optimised conditions. All fluorescent data was acquired to the SYBR channel at the end of each cycle. The amplification and melt curves were visualised using CFX Manager software v. 3.0 (BioRad). The difference curves for HRM were created and analysed using the Precision Melt Analysis software v. 1.2 (BioRad).

A salting out method was used for the extraction of genomic DNA from 400 μl of peripheral blood. DNA quantification was carried out in a Qubit 2.0 fluorometer (Thermo Fisher Scientific, MA, USA) using the Qubit dsDNA BR assay kit (Thermo Fisher Scientific) and then normalized to 10 ng/μl and stored at 4 °C until used. Genotyping of the rs12720208 SNP in the FGF20 gene was made using the TaqMan SNP genotyping assay (Thermo Fisher Scientific). For the qPCR reaction, it was used 5 μl of TaqMan genotyping Master Mix (Thermo Fisher Scientific), 0.5 μl of the pre-design TaqMan probe (c__31674955_10, Thermo Fisher Scientific), 20 ng of genomic DNA and ultra-pure water, for a final volume of 10 μl. The standardized PCR program for TaqMan assays was performed in a CFX96 Touch Real-Time PCR system (BioRad, Hercules, CA, USA). Genotyping results were analyzed with the CFX Manager Software v.3.0 (BioRad). A random 10 % of the samples was reanalyzed for genotyping confirmation.

Total RNA was extracted and purified as described above. cDNA was synthesized using the iScript cDNA Synthesis Kit (BioRad). Reverse transcription quantitative PCR was performed on a Bio-Rad CFX Connect Real-Time System using SsoAdvanced SYBR Green Supermix (Bio-Rad) with a two-step cycling protocol and annealing/extension temperature of 58.5°C. At least three biological replicates and two technical replicates were performed for each experiment. The relative amount of each cDNA target was normalized to Smed-β-tubulin (accession no. DN305397). The normalized relative changes in gene expression, standard deviations, and t-tests were calculated in Bio-Rad CFX Manager Software v3.0. Primers are listed in Table S3.

Relative quantitation real-time PCR was performed on a CFX Connect Real-Time PCR System (Bio-Rad Laboratories). In addition to SsoAdvanced Universal SYBR Green Supermix (Bio-Rad Laboratories) and nuclease-free water, each reaction contained 750 nM of the forward and the reverse primer and 2 µL of cDNA, used at up to 1:10 dilution. Cycling conditions included a pre-amplification hold of 95 °C for 30 s, followed by 40 cycles of denaturation at 95 °C for 30 s, annealing at 60 °C for 30 s, and extension and fluorescence reading at 72 °C for 30 s. Melting curves from 70 °C to 95 °C were performed for each run to confirm a single peak was produced for all primer sets, and amplicon sizes were confirmed by agarose gel electrophoresis. Relative expression of the transcripts of interest, normalized to stable reference genes (coefficient of variation less than 0.25)—5′-aminolevulinate synthase 1 (ALAS1), peptidylprolyl isomerase A (PPIA), or ribosomal protein lateral stalk subunit 0 (RPLP0)—was determined by the Pfaffl method [56 (link)]. Primer efficiency was greater than 85% for all primer sets, as determined by CFX Manager software v3.0 (Bio-Rad Laboratories) using standard curves generated by serial dilution of purified PCR products. Primer sequences and expected product sizes are shown in Table 3.