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 ManagerTM software (Bio-Rad Laboratories). For each DNA sample, three technical replicates were analyzed. Fungal biomass was expressed as ng DNA/μg decayed apple tissue.
Cfx managertm software
Manufactured by Bio-Rad
Sourced in United States
The CFX ManagerTM software is a comprehensive data analysis and reporting tool designed for use with Bio-Rad's real-time PCR detection systems. The software provides users with the necessary functionalities to analyze, interpret, and manage their real-time PCR data.
Automatically generated - may contain errors
Lab products found in correlation
Iqtm sybr green supermix, by Bio-Rad (4 mentions)
Taqman probe, by Thermo Fisher Scientific (4 mentions)
Dnase 1, by Thermo Fisher Scientific (4 mentions)
Cfx96tm real time pcr detection system, by Bio-Rad (3 mentions)
Cfx96 touch real time pcr detection system, by Bio-Rad (3 mentions)
Trizol, by Thermo Fisher Scientific (3 mentions)
Sigmaplot, by Grafiti LLC (2 mentions)
Ssofasttm evagreen supermix, by Bio-Rad (2 mentions)
Iq sybr green supermix kit, by Bio-Rad (2 mentions)
Rnaiso, by Takara Bio (2 mentions)
Iq thermal cycler, by Bio-Rad (2 mentions)
Random hexamer priming kit, by Thermo Fisher Scientific (2 mentions)
Miniopticon real time pcr detection system, by Bio-Rad (2 mentions)
Cfx384 touchtm real time pcr detection system, by Bio-Rad (2 mentions)
Sybr premix, by Takara Bio (2 mentions)
Cfx connecttm real time pcr detection system, by Bio-Rad (2 mentions)
Cfx96tm real time system, by Bio-Rad (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Maxima first strand cdna synthesis kit, by Thermo Fisher Scientific (2 mentions)
Sybr premix ex taqtm 2 tli rnaseh plus, by Takara Bio (1 mentions)
36 protocols using cfx managertm software
1
Quantifying Fungal Biomass in Infected Apples
2
Thermal Shift Assay for MtOPRT Protein
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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 ).
3
Quantitative Analysis of EMT Markers
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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.InTM (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 SSOfastTM 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 ManagerTM 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 program67 (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.
4
Granulosa Cell RNA Extraction and qPCR Analysis
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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 ManagerTM 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.
5
Hypothalamic RNA Isolation and qRT-PCR
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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 .
6
Quantitative Analysis of Gene Expression
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Total RNA was isolated using the PureZOLTM 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 iScriptTM cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA) according to the supplier’s instructions, and was amplified using iQTM 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.
7
Quantifying Gene Expression in Cultured Arteries
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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
8
Identifying Optimal Reference Genes for qRT-PCR Analysis
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The Cq average values calculated for the candidate reference genes in CFX ManagerTM software (Bio-Rad) were analyzed by three different descriptive statistic tools freely available online: NormFinder software (http://moma.dk/normfinder-software ), BestKeeper software (http://www.gene-quantification.de/bestkeeper.html ) and RefFinder web-based tool (http://www.leonxie.com/referencegene.php ). NormFinder calculates not only the overall variation of the candidate normalization genes but also the variation between sample subgroups of the sample set54 (link). The software BestKeeper estimates the correlation of expression levels of all candidates and the genes more stable are combined in an index. Three main indicators were used to determinate the best candidate: standard deviation, covariance and correlation analysis55 . RefFinder ranks all candidates of reference genes based on the main algorithms currently used, including Normfinder and BestKeeper, as well as the geNorm algorithm56 and the Comparative ΔCt Method57 (link). Thus, RefFinder allows the choice of the best candidates based on the final ranking results of different programs58 (link)59 (link)60 (link)61 (link)62 (link).
9
Validating RNA-seq Findings by RT-qPCR
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Findings from the RNA-sequencing data analysis were validated in the same RNA samples by real-time quantitative PCR (RT-qPCR). To this end, first-strand cDNA was synthesized with the RevertAid H Minus First Strand cDNA synthesis kit using random hexamer primers (Thermo Scientific Fermentas, Vilnius, Lithuania). RT-qPCR experiments were carried out in duplicate on the CFX384 Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA) using iQ™ SYBR® Green (Bio-Rad) as fluorophore and exon-spanning primers (Supplementary Table 2 ). TBP was selected as reference gene based on its low variability in the RNA-seq data and high stability in RT-qPCR analysis. The RT-qPCR data was analysed with Bio-Rad CFX ManagerTM Software (version 3.1). Statistical analysis was performed using a linear model, correcting for age, gender, current smoking status and leukocyte counts (basophils, eosinophils, lymphocytes, monocytes and neutrophils).
10
Quantifying Malaria Parasite Burden in Mice
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For parasite burden detection, the livers of mice were dissected at 46 h after infection with CSPwt or CSPmut sporozoites, and homogenized in 1.5 mL Trizol (Invitrogen), and HepG2 cells were collected 46 h after infection with CSPwt or CSPmut sporozoites and lysed with 1 mL Trizol (Invitrogen). Liver homogenate (100 μL) or 1 mL of cell lysis were used for total RNA extraction using Trizol in accordance with the manufacturer’s instructions. cDNA was synthesized from equivalent total RNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (TAKARA) in accordance with the manufacturer’s instructions. The parasite load in the livers and HepG2 cells was evaluated by Taqman PCR with primers and probes for 18S rRNA and GAPDH following the manufacturer’s instructions of Premix Ex Taq™ (Probe qPCR) (TAKARA). The mRNA levels of IFN-γ, iNOS, LC3B, ATG3, ATG5, ATG7, STUB1, SYVN1, CBL, SMURF1, PAFAH1B1, and NEDD4 were evaluated using TB Green™ Premix Ex Taq™ II (Tli RNaseH Plus) (TAKARA). CFX96 Touch™ Real-Time PCR Detection System and CFX ManagerTM Software (Bio-Rad, Hercules, CA, USA) were used for RT-PCR data collection and analysis. The annealing temperature used for the above two RT-qPCR experiments was 60°C.
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