Biorad c1000 thermal cycler cfx96 real time system

Manufactured by Bio-Rad

Sourced in United States, Germany

The BioRad C1000 Thermal Cycler/CFX96 Real-Time System is a laboratory equipment designed for DNA amplification and real-time quantitative PCR analysis. It features a thermal cycler for running polymerase chain reaction (PCR) experiments and a real-time detection system for quantifying nucleic acid targets.

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Lab products found in correlation

Sybr green pcr master mix, by Promega (2 mentions) Sybr green kit, by Promega (2 mentions) Rt2 sybr green mastermix, by Qiagen (1 mentions) Rt2 profiler pcr array, by Qiagen (1 mentions) Sybr premix ex taq perfect real time, by Takara Bio (1 mentions) Cfx manager v1, by Bio-Rad (1 mentions) Sybr premix ex taq, by Takara Bio (1 mentions) Primescript rt reagent kit with gdna eraser, by Takara Bio (1 mentions) Nucleospin rna plus columns, by Macherey-Nagel (1 mentions) Sensifast cdna synthesis kit, by Meridian Bioscience (1 mentions) Il 10 mouse uncoated elisa kit, by Thermo Fisher Scientific (1 mentions) Sensifast sybr hi rox kit, by Meridian Bioscience (1 mentions) Tri reagent, by Merck Group (1 mentions) Cfx manager software, by Bio-Rad (1 mentions) Rneasy kit, by Thermo Fisher Scientific (1 mentions) Sybr green, by Thermo Fisher Scientific (1 mentions) Superscript 2 reverse transcriptase, by Thermo Fisher Scientific (1 mentions) Sypro orange fluorescent dye, by Thermo Fisher Scientific (1 mentions) Matlab r2014a, by MathWorks (1 mentions) Taqman snp genotyping assay, by Thermo Fisher Scientific (1 mentions)

10 protocols using biorad c1000 thermal cycler cfx96 real time system

TGF-β/Smad Signaling Gene Expression Profiling

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The RT² Profiler PCR Array (Qiagen, Hilden, Germany) was used to profile the expression of 84 key genes involved in TGF-β/Smad-related components.
Synthesis of the cDNA was performed with the RT² first strand kit (Siegen) according to the manufacturer's instructions at 42°C for 15 min with a 5-min deactivation step at 95°C in a BioRad CFX96 Real-Time System C1000 Thermal Cycler (Biorad, Munic, Germany).
The RT² SYBR green master mix (Qiagen) (1350 μl per 96-well plate) was mixed with 1248 μl RNase free water and 102 μl cDNA synthesis reaction template, and 25 μl PCR components were added to each well of the array. Quantitative real time polymerase chain reaction (qRT-PCR) was performed in accordance with the recommendations of the manufacturer. Cycling and detection were done in a BioRad CFX96 Real-Time System C1000 Thermal Cycler (Biorad, Munic, Germany).

Wu R.S., Hong J.J., Wu J.F., Yan S., Wu D., Liu N., Liu Q.F., Wu Q.W., Xie Y.Y., Liu Y.J., Zheng Z.Z., Chan E.C., Zhang Z.M, & Li B.A. (2017). OVOL2 antagonizes TGF-β signaling to regulate epithelial to mesenchymal transition during mammary tumor metastasis. Oncotarget, 8(24), 39401-39416.

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Relative Expression Analysis of RrNHX1 and RrVHA-c

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mRNA relative expression of RrNHX1 and RrVHA-c was analyzed by the method of real-time quantitative RT-PCR with a BIO-RAD CFX96™ Real-Time System (C1000™ Thermal Cycler) (Bio-Rad, USA). cDNA was synthesized from 1 μg RNA using PrimeScript® RT reagent Kit with gDNA Eraser (TaKaRa, Japan). Rosa hybrid α-tubulin subunit actin gene (GenBank Accession No. AF394915.1) was used as an internal constitutively expressed control in the real-time quantitative RT-PCR analysis. PCR was performed using the primers shown in Table 2. Quantitative real-time PCR experiments were performed according to the instructions of the SYBR® Premix Ex Taq™ (Perfect Real Time) (TaKaRa, Japan) and contained 2× SYBR Premix Ex Taq™ 12.5 μl, 50× ROX Reference Dye II 0.5 μl, 2 μl cDNA solution as a template, 1 μl mix solution of target gene primers and 9 μl ddH₂O in a total volume of 25 μl. The amplification was performed by an initial incubation at 50 °C for 2 min and at 95 °C for 5 min, then followed by 40 cycles of 15 s at 95 °C, 15 s at 51 °C, and 40 s at 72 °C. Expression levels for each gene were calculated by the 2^−△△Ct comparative threshold cycle (Ct) method (Schmittgen and Livak, 2008 (link)). The Ct values were generated from the Bio-Rad CFX Manager V1.6.541.1028 software. All samples were repeated three times.

Feng L., Ding H., Wang J., Wang M., Xia W., Zang S, & Sheng L. (2015). Molecular cloning and expression analysis of RrNHX1 and RrVHA-c genes related to salt tolerance in wild Rosa rugosa. Saudi Journal of Biological Sciences, 22(4), 417-423.

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Quantification of PD-L1 and RIP2 Expression

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The cells were harvested after 24h for ribonucleic acid (RNA) extraction. The RNA was extracted using the Nucleo Spin^® RNA Plus columns and solutions following the manufacturer´s instructions (Machery-Nagel, Dueren, Germany). Reverse transcription was performed with the Verso cDNA sythesis kit (ThermoFisher) following the manufacturer´s protocol. For quantitative real time (RT) PCR the SensiFAST ™SYBR No-ROX Mix was used (Bio Cat, Heiderberg, Germany). As house-keeping reference gene glycerinaldehyd-3-phosphat-dehydrogenase (GAPDH) was used. As primer the Quantitect Primer assay Hs_CD274_1_SG (PD-L1), Hs_RIPK1_1_SG (RIP2) and Hs_GAPDH_1_SG (GAPDH) were chosen (Qiagen, Hilden, Germany).
PCR runs and detection were performed in a BioRad CFX96 Real-Time System C1000 Thermal Cycler (BioRad, Feldkirchen, Germany).
The outcome was analyzed using the ΔΔ CT method. The results are shown normalized to reference and relative to non-treated negative control

Groeger S., Wu F., Wagenlehner F., Dansranjav T., Ruf S., Denter F, & Meyle J. (2022). PD-L1 Up-Regulation in Prostate Cancer Cells by Porphyromonas gingivalis. Frontiers in Cellular and Infection Microbiology, 12, 935806.

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Quantification of IL-10 levels

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ALT and AST levels were quantified in the serum using an AutoAnalyzer (Prestige 24i, PZ Cormay S.A) as previously described [10] (link).
2.8. IL-10 quantification IL-10 levels were quantified on blood, spleen and liver homogenates by ELISA and the transcriptional levels of Il10 gene were assessed by real-Time quantitative PCR (qRT-PCR). Briefly, spleen and liver were homogenized and stored for IL-10 quantification using IL-10 mouse uncoated ELISA kit (ThermoScientific) or in TRIreagent (Sigma-Aldrich) for RNA extraction. Total RNA was extracted and the synthesis of cDNA was made with SensiFAST™ cDNA Synthesis Kit (Bioline). qRT-PCR reactions were run for each sample on a Bio-Rad CFX96 Real-Time System C1000 Thermal Cycler (Bio-rad) using SensiFAST SYBR Hi-ROX Kit (Bioline). Primer sequences were obtained from Alfagene (Portugal) and thoroughly tested. The results were normalized to the expression of the housekeeping gene ubiquitin. After amplification, cycle thresholdvalues (Ct-values) were calculated for all samples and gene expression changes were analyzed in the CFX Manager Software (Bio-Rad). The serum, splenic and liver IL-10 transcript and expression levels are shown on Supplementary figure 2A and B respectively.

Mesquita I., Ferreira C., Barbosa A.M., Ferreira C.M., Moreira D., Carvalho A., Cunha C., Rodrigues F., Dinis-Oliveira R.J., Estaquier J., Castro A.G., Torrado E, & Silvestre R. (2018). The impact of IL-10 dynamic modulation on host immune response against visceral leishmaniasis. Cytokine, 112.

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Quantitative PCR Analysis of Key Genes

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The total RNA isolation and cDNA synthesis were performed with commercial kits (Qiagen, Germany). Specific primers and probes for VEGFA, E-cadherin, EGFR, TGFβ1, bFGF and β-actin were designed and checked with Primer3web (version 4.0.0), Primer-Blast, NCBI-Blast. The primers and TaqMan probes used for qPCR are listed in Supplementary Table 1. The mRNA expression by qPCR was repeated thrice for each cell line. The qPCR amplification was performed using a 1X qPCR Mix (Solis Biodyne, Estonia). The qPCR profile consisted of 10 min initial denaturation at 95 °C, followed by 40 cycles (95 °C for 20 s, 60 °C for 1 min). qPCR was conducted on the Bio-Rad C1000 Thermal Cycler CFX96 Real-Time System (Bio-Rad, USA). Delta-delta Ct method (2^−ΔΔCt method) was used for gene expression analysis in cells.

BABAHAN C., ABDI ABGARMI S., SONUGÜR F.G., ÖÇAL M, & AKBULUT H. (2023). The effects of anti-PD-L1 monoclonal antibody on the expression of angiogenesis and invasion-related genes. Turkish Journal of Biology, 47(4), 262-275.

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RNA Extraction and qPCR Analysis

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Total RNA was extracted by using RNeasy Kit (ThermoFisher, K0731) according manufacturer's protocol and was reversely transcribed to cDNA using SuperScript Ⅱ reverse transcriptase (Invitrogen, 18064071) with random oligos (Invitrogen, 48190011). The quantitative PCR reaction was carried out with the mixture of SYBR Green (Applied Biosystems, A25743). Real-time PCR amplification and data analysis were performed using BioRad C1000 Thermal Cycler CFX96 Real-Time System (BioRad).

Kong Y., Wu M., Wan X., Sun M., Zhang Y., Wu Z., Li C., Liang X., Gao L., Ma C, & Yue X. (2023). Lipophagy-mediated cholesterol synthesis inhibition is required for the survival of hepatocellular carcinoma under glutamine deprivation. Redox Biology, 63, 102732.

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Quantification of Immune Markers in Cells

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For quantitative analysis, primers specific for Foxp3, PD-1, PD-L1, perforin, granzyme B, and GAPDH were used in real-time PCR with the SYBR Green kit (Promega, USA) in a BioRad C1000 Thermal cycler/CFX96 real-time System (BioRad, Germany). Briefly, 5 ng cDNA was added to a final volume of 10 µl/reaction containing 1 × SYBR Green PCR Master Mix (Promega, USA) and 100 nM of each primer. Thermal cycling conditions were: denaturation at 95˚C 2 min, 42 cycles: 95 °C/10 s, 59 °C/15 s and 72 °C/30 s for elongation. Primers: GAPDH: forward 5′-CCACATCGCTCAGACACCAT-3′ and reverse 5′-GGCAACAATATCCACTTTACCAGACT-3′. Foxp3: forward 5′-GAGAAGCTGAGTGCCATGCA-3′ and reverse 5′-GGAGCCCTTGTCGGATGAT-3′. PD-1: forward 5′-ATCAAAGAGAGCCTGCGGG-3′ and reverse 5′-GGTGGGCTGTGGGCACT-3′. PD-L1: forward 5′-AAATGGAACCTGGCGAAAGC-3′ and reverse 5′-GATGAGCCCCTCAGGCATTT-3′. Granzyme B: forward 5′- TTCGTGCTGACAGCTGCTCACT-3′ and reverse 5′- CTCTCCAGCTGCAGTAGCATGA-3′. Perforin: forward 5′-ACCAGCAATGTGCATGTGTCTG-3′ and reverse 5′- GCCCTCTTGAAGTCAGGGT-3’.

Schilbach K., Krickeberg N., Kaißer C., Mingram S., Kind J., Siegers G.M, & Hashimoto H. (2020). Suppressive activity of Vδ2+ γδ T cells on αβ T cells is licensed by TCR signaling and correlates with signal strength. Cancer Immunology, Immunotherapy, 69(4), 593-610.

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Thermal Stability Analysis of FLN Fragments

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Thermal stabilities of the WT and mutated FLN fragments were determined using Bio-Rad C1000 Thermal Cycler, CFx96 Real-Time system (Bio-Rad Laboratories) with SYPRO Orange fluorescent dye (Invitrogen)^{73 (link)}. A temperature gradient from 20 to 95 °C was used with 0.5 °C increment every 30 seconds. Samples contained 100–200 μM protein and 5 X SYPRO Orange dye in total volume of 25 μl. The data was collected from three individual experiments and plotted using MATLAB R2014a (The MathWorks, Inc.).

Seppälä J., Bernardi R.C., Haataja T.J., Hellman M., Pentikäinen O.T., Schulten K., Permi P., Ylänne J, & Pentikäinen U. (2017). Skeletal Dysplasia Mutations Effect on Human Filamins’ Structure and Mechanosensing. Scientific Reports, 7, 4218.

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Genotyping PDYN Promoter SNP rs1997794

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DNA was purified from human brain samples using Wizard Genomic DNA Purification kit (Promega, Madison, USA). Genotyping of SNP rs1997794 located in PDYN promoter was performed by allelic discrimination using TaqMan SNP Genotyping Assay C_11670951_10 (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's protocol. Polymerase chain reactions were set up in a total volume of 10 μl, including 1 × iTaq Universal Probes Supermix (Bio-Rad, Sunnyvale, CA, USA), 1 × TaqMan SNP Genotyping Assay (Applied Biosystems), and 10 ng of template DNA using the BioRad C1000 Thermal Cycler (CFX96 Real-Time System) (Bio-Rad). After an initial denaturation step for 10 min at 95°C, each cycle consisted of denaturation for 15 s at 95°C and annealing and primer extension for 60 s at 62°C for a total 40 cycles. The rs1997794 variant pattern was set up with DNA from positive controls previously genotyped (Taqi et al., 2011 (link)).

Sarkisyan D., Hussain M.Z., Watanabe H., Kononenko O., Bazov I., Zhou X., Yamskova O., Krishtal O., Karpyak V.M., Yakovleva T, & Bakalkin G. (2015). Downregulation of the endogenous opioid peptides in the dorsal striatum of human alcoholics. Frontiers in Cellular Neuroscience, 9, 187.

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Quantitative Analysis of Immune Markers

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For (quantitative) analysis of cytokine and transcription factor expression, perforin-, granzyme B-, class I-restricted T cell-associated molecule (CRTAM)- and GAPDH-specific primers were used in combination with the SYBR Green kit (Promega, USA) in a BioRad C1000 Thermal cycler/CFX96 real-time System (BioRad, Germany). GAPDH was used as reference gene. Briefly, cDNA was added to a final volume of 10 μl/reaction containing 1 × SYBR Green PCR Master Mix (Promega, USA) and 100 nM of each primer. Thermal cycling conditions were: denaturation at 95 °C 10 min, 40 cycles: 95 °C/30′′, 60 °C/30′′ and 72 °C, 1 min for elongation. Primer sequences were: CRTAM-for: 5′-CCTCTCAAGACCCACAGCAG-3′, CRTAM-rev: 5′-AATGAGGAA-GGACACCAGCG-3′, perforin-for, 5′-ACCAGCAATGTGCATGTGTCTG-3′ and perforin–rev: 5′-GGCCCTCTTGAAGTCAGGGT-3′ [37 (link)], GrzB for: 5′-TTCGTGCTGA-CAGCTGCTCACT-3′ and GrzB-rev, 5′-CTCTCCAGCTGCAGTAGCA-TGA-3′ [38 (link)], GAPDH-for: 5′-CCACATCGCTCAGACACCAT-3′ and GAPDH-rev: 5′-GGCAACAA-TATCCACTTTACCAGACT-3′ (RTPrimerDB ID 2053).

Sonntag K., Hashimoto H., Eyrich M., Menzel M., Schubach M., Döcker D., Battke F., Courage C., Lambertz H., Handgretinger R., Biskup S, & Schilbach K. (2018). Immune monitoring and TCR sequencing of CD4 T cells in a long term responsive patient with metastasized pancreatic ductal carcinoma treated with individualized, neoepitope-derived multipeptide vaccines: a case report. Journal of Translational Medicine, 16, 23.

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