Cfx manager software

qPCR assays with DNA samples extracted from infected apples were performed to determine fungal biomass using SYBR Premix Ex Taq TM II (Tli RNaseH Plus) (TAKARA, Japan) with the Bio-Rad CFX96 (Bio-Rad, Hercules, CA, United States) and the primer pair patF_F and/patF_R. Two negative controls were also performed (one without primers and fungal DNA and the other one without fungal DNA) to rule out any possible matrix effect. The PCR conditions were as follows: 94°C for 30 s, 40 cycles of 94°C for 20 s, 60°C for 20 s, and 72°C for 20 s. The dissociation curve analysis followed the same trend as the amplification cycle and was constructed by continuously measuring the fluorescence when increasing the temperature from 65 to 95°C, at the rate of 0.5°C/s. The threshold cycle (CT) values were automatically determined by the CFX Manager^TM software (Bio-Rad Laboratories). For each DNA sample, three technical replicates were analyzed. Fungal biomass was expressed as ng DNA/μg decayed apple tissue.

The reaction mix used in the thermal shift assay consisted of the MtOPRT protein diluted to a final concentration of 0.5 mg/mL and with the fluorescence probe SYPRO Orange (Sigma–Aldrich) at 1/4000 dilution. The final reaction volume was 20 µl. Fluorescence was recorded in a 48-well plate using the FAM channel of a MiniOpticonTM Real-Time PCR Detection System (Bio-Rad). Data were harvested and analyzed using CFX ManagerTM Software (Bio-Rad) and SigmaPlot (Systat Software, San Jose, CA, SigmaPlot.com">www.SigmaPlot.com).

Total RNA was isolated using Trizol® (Life Technologies), and 5 µg of total RNA was treated with DNase I (Life Technologies), in the presence of SUPERase^.In^TM (Life Technologies) for 30 minutes at 37 °C; EDTA was then added to 5 mM and the DNase I heat inactivated at 75 °C for 10 minutes. After adding MgCl₂ to 5 mM, cDNA was generated using Superscript III (Life Technologies) and oligo dT following the manufacturer’s instructions. Real time PCR was carried out using SSOfast^TM EvaGreen® Supermix (Bio-Rad, Hercules, CA, U.S.A.), and relative expression levels of EMT marker genes CDH1 (encoding E-cadherin), CDH2 (encoding n-cadherin) and vim (encoding vimentin) determined with the Bio-Rad CFX Manager^TM software, using the reference genes glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glucose-6-phosphate isomerase (G6PI) and small nuclear ribonucleoprotein D3 (SNRPD3). Primers were designed using the Primer 3 program^{67 (link)}. Primer efficiencies were calculated and input into the software. Primer sequences and efficiencies are listed in Table S1. The gene expression was normalised to the two housekeeping genes, and the gene expression changes calculated using the ∆∆CT method as the efficiencies of the primers were all similar. The gene expression changes were displayed as a fold change compared to the control set at a value of 1 unless otherwise stated.

Total RNA was extracted from granulosa cells using the RNAiso Plus kit (Takara, Dalian, China) following the manufacturer’s protocol. Reverse transcription to obtain cDNA was performed using a PrimeScript™ RT reagent kit with a gDNA Eraser (Takara). Primers used in this experiment were synthesized in BGI Company (Shenzhen, China) (Table 1). qPCR detection and expression analysis of genes was then carried out using the iQ SYBR Green Supermix kit (Bio-Rad Laboratories, CA, USA). Threshold and threshold cycle (Ct) values were determined automatically by the CFX Manager^TM Software (Bio-Rad Laboratories) using default parameters. The comparative cycle threshold (2^-ΔΔCt method) was used to analyze the expression levels of genes examined in this study. The abundance of each gene transcripts was normalized by GAPDH gene expression levels and expressed as arbitrary units (AU). The relative quantization of gene expression was performed in three replicates for each sample.

RNA of the hypothalamus was isolated using RNAiso (Takara, Tokyo, Japan) according to the manufacturer’s instructions. The extracted RNA was synthesized with cDNA using a PrimeScript first-strand cDNA Synthesis Kit (Takara). qRT-PCR was performed using cDNA synthesized using the CFX384 TouchTM Real-Time PCR detection system. cDNA (300 ng), SYBR premix (5 μL; Takara), forward primer (0.4 μM), and reverse primer (0.4 μM) were mixed, and the number of threshold cycle were determined using CFX ManagerTM software (BioRad, CA, USA). All primer information is summarized in the Table S2.

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.

Total RNA was extracted from cultured artery and cDNA obtained by reverse transcription using a random hexamer priming kit (Thermo Fisher Scientific, Applied Bioscience) in a final volume of 100 µL. Then, specific pre-developed TaqMan probes from Thermo Fisher Scientific (TaqMan Gene Expression Assays) were used for PCR amplification (listed in Supplementary Table 2). Fluorescence was detected with the CFX96TM Real-Time PCR Detection System and the results analyzed with the CFX ManagerTM software (Bio-Rad Laboratories). Gene expression was normalized to the expression of the endogenous control GUSB using the comparative ΔCt method. mRNA concentration was expressed in relative units with respect to GUSB expression (relative expression).