Cfx manager software version 2

Manufactured by Bio-Rad

Sourced in United States

The CFX Manager Software Version 2.1 is a data analysis software designed for use with Bio-Rad's real-time PCR detection systems. It provides tools for data analysis, result interpretation, and report generation.

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39 protocols using cfx manager software version 2

Quantitative Gene Expression Analysis in Maize Anthers

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B73 maize anthers were staged, dissected and stored in RNAlater. Immediately before processing, anthers were washed in 1xPBS and then used for RNA extraction using the Qiagen RNeasy® Mini Kit. DNAse digest was carried out with Optizyme rDNAseI (Fisher Scientific). ~2.5 μg total RNA per sample as measured with Qubit® RNA BR Assay Kit (Invitrogen) was used for cDNA synthesis with the SuperScript® III First Strand Synthesis System for RT-PCR (Invitrogen) including oligo dT primer. For real-time PCR, iQ™ SYBR® Green Supermix (Bio-Rad) was used with cDNA from ~125 ng total RNA and 25 μl end volume per reaction, performed in triplicates. Primers used are listed in Additional file 12: Table S6. Primer efficiencies were calculated with Real-time PCR Miner [79 (link)], http://www.miner.ewindup.info/ and were in the range of 90-110% per gene. A Bio-Rad real-time PCR system (C100TM Thermal Cycler, with CFX96™ Real-Time System) was used in conjunction with the Bio-Rad CFX Manager™ Software, Version 2.0.

Dukowic-Schulze S., Sundararajan A., Mudge J., Ramaraj T., Farmer A.D., Wang M., Sun Q., Pillardy J., Kianian S., Retzel E.F., Pawlowski W.P, & Chen C. (2014). The transcriptome landscape of early maize meiosis. BMC Plant Biology, 14, 118.

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Quantification of Microbial Transcripts in Dental Samples

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Total bacterial RNA from supragingival samples (collected in the first recruitment, n = 15 from each group) were isolated and purified according to the manufacturer’s instructions of RNeasy Mini Kit (Qiagen, Valencia, CA, USA), and reverse transcribed (500 ng RNA) using Prime Script RT Reagent Kit (TaKaRa, Japan) with random hexamer primers. qPCR amplification was performed on the CFX96 system (Bio-Rad, Hercules, CA, USA). The reaction mixture (25 μl) contained 1 × SYBR Premix Ex Taq (TaKaRa), 2 μl template cDNA, forward and reverse primers (500 nM each; sequences were listed in Supplementary Table S4). Expression level alterations of arcA, ureC and ldh after arginine-dentifrice treatment were calculated by Bio-Rad CFX Manager software (version 2.0) according to the 2^{−∆∆ CT} method.

Zheng X., He J., Wang L., Zhou S., Peng X., Huang S., Zheng L., Cheng L., Hao Y., Li J., Xu J., Xu X, & Zhou X. (2017). Ecological Effect of Arginine on Oral Microbiota. Scientific Reports, 7, 7206.

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Gene Expression Analysis of Inflammatory Markers

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Total RNA was isolated from either kidney tissues or cultured cells using Tri reagent (Molecular Research Center, Inc, Cincinnati, OH, USA) per manufacturer's protocol. Single-strand cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) for two-step qRT-PCR. Quantitative PCR was performed using Taqman gene expression assays (TNF-α: Mm00443260_g1, CCL2/MCP-1: Mm00441242_m1, TNFR1: Mm00441883_g1, TNFR2: Mm00441889_m1, GAPDH: Mm99999915_g1; Life Technologies, Grand Island, NY, USA) in a Bio-Rad CFX96 Real-Time System. Data were analyzed using Bio-Rad CFX Manager Software version 2.0. Relative expression quantification was calculated using the 2^(-ΔΔCT) equation after normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously.^{15 (link),73}

Awad A.S., You H., Gao T., Cooper T.K., Nedospasov S.A., Vacher J., Wilkinson P.F., Farrell F.X, & Reeves W.B. (2015). Macrophage-derived Tumor Necrosis Factor-α mediates diabetic renal injury. Kidney international, 88(4), 722-733.

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Quantitative Real-Time PCR Analysis

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Primers used have been described previously (13) (link). Quantitative real-time polymerase chain reaction was performed on the CFX 96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, California). The cycle threshold (Ct) value determined with CFX Manager Software version 2.0 (Bio-Rad) for all samples was normalized to Gapdh, and the relative fold change was computed by the comparative Ct (ΔΔCt) method.

Ikeda S., Mukai R., Mizushima W., Zhai P., Oka S.I., Nakamura M., Del Re D.P., Sciarretta S., Hsu C.P., Shimokawa H, & Sadoshima J. (2019). Yes-Associated Protein (YAP) Facilitates Pressure Overload–Induced Dysfunction in the Diabetic Heart. JACC: Basic to Translational Science, 4(5), 611-622.

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RNA Extraction and Gene Expression Analysis in Olive Flounder

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The tissue fragments of head kidney (HK) that were collected from the dissected fish were immediately preserved at −80 °C in TRIzol reagent (Thermo Fisher Scientific, San Jose, CA, USA) for RNA extraction. Total RNA was extracted from 0.5 g of olive flounder tissue using TRIzol reagent (Thermo Fisher Scientific, San Jose, CA, USA). Moreover, fish tissues were analyzed after quantitative analysis and purity assessment using a microvolume UV-Vis spectrophotometer (NanoDrop One, Thermo Fisher Scientific, San Jose, CA, USA). Then, the DNase I enzyme (Cosmogenetech, Seoul, Rep. of Korea) was mixed with the isolated RNA from tissues to exclude genomic DNA from the samples. Afterwards, we used the M-MuLV reverse transcriptase (Cosmogenetech) to produce complementary DNA (cDNA) from the samples. The qRT-PCR analyzer (Bio-Rad CFX96, Bio-Rad, Hercules, CA, USA) was then run with SYBR-Green reagent to determine the expression levels of the four selected immune-related genes such as FGH (flounder growth hormone), IL-1β (interleukin 1β), IL-10 (Interleukin 10), and β-actin (beta-actin as house-keeping gene) which were calculated by using CFX software in triplicates (CFX manager software version 2.0, Bio-Rad) (Table 2).

Choi W., Moniruzzaman M., Bae J., Hamidoghli A., Lee S., Choi Y.H., Min T, & Bai S.C. (2022). Evaluation of Dietary Probiotic Bacteria and Processed Yeast (GroPro-Aqua) as the Alternative of Antibiotics in Juvenile Olive Flounder Paralichthys olivaceus. Antibiotics, 11(2), 129.

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Gene Expression Analysis of Inflammatory Markers

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Quantitative Analysis of AnSnRK2s

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A set of specific primers of AnSnRK2s and a pair of specific primers of AnActin (GenBank accession number: KJ873129) for the internal control were designed by Primer5.0 and synthesized at Sangon (China) (Table S2). The qRT-PCR was performed using SYBR Green Ⅰ kit (TAKARA, Dalian) in CFX-96 system (Bio-Rad, Hercules, CA, USA) as described by Yu et al. [57 (link)]. The 2^–ΔΔCT method of the CFX Manager software version 2.0 (Bio-Rad, USA) was used to normalize the expression differentiation between the internal control and the AnSnRK2s [58 (link)]. The data are presented as the mean values ± standard deviation (SD). The statistical significance among three biological replicates was tested by Microsoft Excel 2017 and SPSS 17.0 software based on Student’s t-tests.

Tang Y., Lu F., Feng W., Liu Y., Cao Y., Li W., Fu F, & Yu H. (2021). Genome-Wide Identification and Expression Analyses of AnSnRK2 Gene Family under Osmotic Stress in Ammopiptanthus nanus. Plants, 10(5), 882.

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Quantitative Analysis of Kidney Gene Expression

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Total RNA was isolated from kidneys using Tri reagent (Molecular Research Center, Inc, Cincinnati, OH, USA) per manufacturer’s protocol. Single-strand cDNA was synthesized using iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA) for two-step qRT-PCR. Quantitative PCR was performed using Taqman gene expression assays (TNF-α: Mm00443260_g1, fibronectin: Mm01256744_m1, VEGFA: Mm01281449_m1, EGFR: Mm00433023_m1; GAPDH: Mm99999915_g1; Life Technologies, Grand Island, NY, USA) using a Bio-Rad CFX96 Real-Time System. Data were analyzed using Bio-Rad CFX Manager Software version 2.0 and relative expression quantified using the 2^(-∆∆CT) equation after normalization to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as described previously [18 (link), 22 (link), 25 (link), 26 (link)].

Awad A.S., You H., Gao T., Gvritishvili A., Cooper T.K, & Tombran-Tink J. (2015). Delayed Treatment with a Small Pigment Epithelium Derived Factor (PEDF) Peptide Prevents the Progression of Diabetic Renal Injury. PLoS ONE, 10(7), e0133777.

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Quantification of Viral RNA in Lungs

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Viral titers of PR8 or PR8-OVA_II-infected lungs were determined by quantification of viral RNA. Whole lungs were homogenized in TRIsol/Chloroform (Sigma-Aldrich) and RNA was extracted using the VWR E.Z.N.A kit and Turbo DNA-free kit (Thermofisher). 2.0μg of RNA was reverse transcribed into cDNA using the High Capacity cDNA reverse transcription kit (Thermofisher). Quantitative PCR was performed to amplify the acidic polymerase (PA) gene using the Bio-Rad CFX96 Realtime PCR system with 50ng of cDNA per reaction. The following primers and probe were used: forward primer: 5′-CGGTCCAAATTCCTGCTGA-3′; reverse primer: 5′CATTGGGTTCCTTCCATCCA-3′; probe: 5′-6-FAM-CCAAGTCATGAAG GAGAGGGAATACCGCT-3′. Data were analyzed using the CFX Manager Software Version 20 (Bio-Rad). A standard curve generated using a PA-containing plasmid obtained from Dr. Rob Webster at St. Jude’s Children’s Research Hospital was used to calculate the PA gene copy number per 50 ng of cDNA. This was used to calculate the total PA copy number per lung.

Bautista B.L., Devarajan P., McKinstry K.K., Strutt T.M., Vong A., Jones M., Kuang Y., Mott D, & Swain S.L. (2016). Short-lived Antigen Recognition but not Viral Infection at a Defined Checkpoint Programs Effector CD4 T Cells to become Protective Memory. Journal of immunology (Baltimore, Md. : 1950), 197(10), 3936-3949.

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Quantifying mRNA Levels in Mice Tissues

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For gene expression analyses, mRNA was obtained from the hypothalamus tissue of adult mice and GT1-7 cells using RNeasy Plus Mini Kits (Qiagen, Chatsworth, CA, US). We used 1000 ng total mRNA as the template for cDNA synthesis primed with random hexamers (Bio-Rad, Richmond, CA, US). Then, the reaction mixture was diluted at 1:2, and 2 μl of the diluted template was used per 20 μl of the real-time quantitative PCR assay using the Bio-Rad SsoAdvanced™ Universal SYBR (Bio-Rad 172–5274, Mississauga, ON, Canada). The Bio-Rad CFX Connect Real-Time system and the Bio-Rad CFX Manager Software (version 2.0) were used to collect the PCR data. Results are presented as linearized values normalized against β-actin. The primers (Table S2) were synthesized by the Beijing Genomic Institute (BGI).

Luo J., Yang Y., Zhang T., Su Z., Yu D., Lin Q., Chen H., Zhang Q., Xiang Q., Xue W., Ge R, & Huang Y. (2018). Nasal delivery of nerve growth factor rescue hypogonadism by up-regulating GnRH and testosterone in aging male mice. EBioMedicine, 35, 295-306.

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