Cfx96 c1000

Manufactured by Bio-Rad

Sourced in United States

The CFX96 C1000 is a real-time PCR detection system designed for quantitative gene expression analysis and genotyping. It features a 96-well format and can accommodate a variety of sample types and reaction volumes. The system utilizes optical detection and thermal cycling capabilities to enable accurate and reliable nucleic acid quantification.

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36 protocols using cfx96 c1000

RNA Extraction and RT-PCR Analysis of Rice

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Total RNA was isolated from various rice tissues using Trizol reagent (Tiangen, Beijing, China), and analyzed with a Nanodrop 1000 spectrophotometer (Thermo Scientific, UT) for assessment of quality and quantity. For RT-PCR, first-strand cDNA was reverse transcribed from total RNA with the PrimeScript™ RT reagent Kit with gDNA Eraser (Perfect Real Time; TaKaRa). Real-time RT-PCR was performed with iQ SYBR Green Supermix (Bio-rad), using the real-time PCR system (Bio-Rad C1000 CFX96). The rice actin gene was used as an internal control. Primers used to quantify the expression of OsTKPR1 are listed in Additional file 7: Table S4.

Xu D., Qu S., Tucker M.R., Zhang D., Liang W, & Shi J. (2019). Ostkpr1 functions in anther cuticle development and pollen wall formation in rice. BMC Plant Biology, 19, 104.

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Quantitative Real-Time PCR Protocol

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Aliquots of total RNA used for sequencing as described earlier were used for qRT-PCR [30 (link)]. cDNA was synthesized and quantified using the Nanodrop (ND-1000) spectrophotometer (Nanodrop products, Wilmington, DE). Twenty-one primer pairs from differential expressed candidate genes were designed using an online PRIMEGENS program (http://primegens.org/) and synthesized from IDT Technologies (Integrated DNA Technologies, Coralville, IA). The working stock of each primer was adjusted to 2 μM. The gene IDs and primer sequences were listed in Table S1 in Supplementary Material available online at http://dx.doi.org/10.1155/2015/528395. Real-time PCR was run on BioRad C-1000 (CFX96) real-time system using the iQ SYBR Green Supermix (BioRad Laboratories, Hercules, CA). For internal control, two housekeeping genes, cab-h1 and cab-h8, from nondifferential expressed genes were used to calculate threshold differences and fold expression differences from differential genes. The abundance levels of the selected transcripts normalized to housekeeping genes were calculated using the 2^−ΔΔCt method [31 (link)]. Before conducting qRT-PCR, regular PCR was performed at an initial denature at 94°C for 3 min, followed by 94°C 40 sec, 58°C 30 sec, and 72°C 40 sec for 20 cycles with a final extension of 3 min at 72°C.

Wang X., Shi W, & Rinehart T. (2015). Transcriptomes That Confer to Plant Defense against Powdery Mildew Disease in Lagerstroemia indica. International Journal of Genomics, 2015, 528395.

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Quantifying IL-10 Expression in Sorted Monocytes

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Monocytes from CD11b enriched cell suspension (Miltenyi) were sorted (FACSAriaIII sorter). Total RNA was extracted using the RNeasy Minikit (Qiagen). Equal amounts of total RNA from each sample were reverse transcribed into cDNA using SuperScript™ VILO™ Master Mix (Invitrogen). qPCR was set up using PowerUp SYBR Green PCR Master Mix (Applied Biosystems) and analyzed on Bio-Rad C-1000 CFX96. RT-PCR primers for IL-10: Forward-GGTTGCCAAGCCTTATCGGA; Reverse-ACCTGCTCCACTGCCTTGCT. Relative quantification of gene expression was performed by normalizing expression levels of genes of interest over housekeeping (Thoc1) gene within each sample followed by relative expression to control (mean of triplicate).

Tewari A., Prabagar M.G., Gibbings S.L., Rawat K, & Jakubzick C.V. (2021). LN Monocytes Limit DC-Poly I:C Induced Cytotoxic T Cell Response via IL-10 and Induction of Suppressor CD4 T Cells. Frontiers in Immunology, 12, 763379.

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Transcriptional Analysis of Osmotic Stress Response

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For transcript analysis during growth during osmotic stress, 1 × 10⁶ conidia of AF293, AF293 ΔsakA, and LH-EVOL strains were grown in a six-well plate in 5 ml of GMM medium for 30 h at 37°C. Mycelia were transferred to a new six-well plate with either GMM or GMM supplemented with 1 M NaCl under sterile conditions and incubated for 30 min at 37°C. Mycelia were transferred to ZR BashingBead Lysis Tubes (0.1 and 0.5 mm; Zymo Research) and homogenized at 4°C using a bead beater for 4 min at 250 rpm. RNA was purified from lysate following the manufacturer’s protocol for Quick-RNA Fungal/Bacterial Miniprep kit (Zymo Research). cDNA was synthesized following the manufacturer’s protocol for QuantiTect reverse transcription kit (Qiagen) including genomic DNA (gDNA) removal. Quantitative PCR was performed using SsoAdvanced Universal SYBR Green Supermix according to the manufacturer’s directions and primers described in Table S3 using the thermocycler Bio-Rad C-1000 CFX96. Cycling parameters were according to the SsoAdvanced Universal SYBR Green Supermix manufacturer’s recommendation using 60°C as the annealing temperature. Transcripts were normalized to actA using the 2^−ΔΔCt method (68 (link)).

Kirkland M.E., Stannard M., Kowalski C.H., Mould D., Caffrey-Carr A., Temple R.M., Ross B.S., Lofgren L.A., Stajich J.E., Cramer R.A, & Obar J.J. (2021). Host Lung Environment Limits Aspergillus fumigatus Germination through an SskA-Dependent Signaling Response. mSphere, 6(6), e00922-21.

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Analysis of Hypothalamic Gene Expression in WT and MT1KO Mice

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Hypothalami of WT and MT₁KO mice were collected at four different time points, immediately frozen, and then stored at −80 °C. mRNA was then isolated using TRIzol® (Invitrogen, Carlsbad, CA, USA) and cDNA was synthesized using the Improm-II- Reverse Transcriptase System (Promega, Madison, WI, USA). Real-time PCR analyses of the ObRb, Npy, AgRP, Pomc and Socs3 genes were performed (for the primer designs, see Table 1) using Power SyBR Green (Applied Biosystems, Waltham, MA, USA) in a Bio-Rad C1000 CFX96. The data were analyzed by 2^−ΔΔCT and normalized by the geometric mean of Rpl and Ppia.

Buonfiglio D., Tchio C., Furigo I., Junior J.D., Baba K., Neto J.C, & Tosini. G. (2019). Removing Melatonin Receptor Type 1 Signaling Leads to Selective Leptin Resistance in The Arcuate Nucleus. Journal of pineal research, 67(2), e12580.

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SARS-CoV-2 nsp3 macrodomain thermal stability

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The SARS-CoV-2 nsp3 macrodomain without tags was diluted to 5 μM in 10 mM HEPES pH 7.5, 25 mM NaCl, 0.5 mM TCEP buffer and mixed with 5x SYPRO Orange. Samples were prepared with 10 μM, 50 μM, 100 μM or 1 mM of suramin. Samples in presence or absence of 1 mM ADP-ribose were used as controls. Samples were transferred to 96-well qPCR plates. Measurement was performed in a BioRad C1000 CFX96 thermal cycler. Data points for melting curves were recorded in 1 min intervals from 20–95°C, with the temperature increasing by 1°C/min. The analysis of the data was done in GraphPad Prism 8 using a nonlinear regression analysis (Boltzmann sigmoid equation) of normalized data.

Sowa S.T., Galera-Prat A., Wazir S., Alanen H.I., Maksimainen M.M, & Lehtiö L. (2021). A molecular toolbox for ADP-ribosyl binding proteins. Cell Reports Methods, 1(8), 100121.

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Quantitative PCR Analysis of Mouse Colon Transcripts

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RNA was extracted from mouse colon tissue using the High Pure RNA Tissue Kit (Roche). RNA was reverse transcribed into cDNA using the cDNA Synthesis Kit (Roche) according to the manufacturer's instructions. Quantitative real-time PCR (pPCR) was performed with SYBR-Green Mastermix (Roche) and the following gene-specific primers (Table 1) on a BioRad C1000/CFX96 real time machine. Data were normalized to house-keeping genes Gapdh and Hprt1 and fold change was calculated using the ΔΔC_t method.

Schramm G., Suwandi A., Galeev A., Sharma S., Braun J., Claes A.K., Braubach P, & Grassl G.A. (2018). Schistosome Eggs Impair Protective Th1/Th17 Immune Responses Against Salmonella Infection. Frontiers in Immunology, 9, 2614.

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Quantifying Viral Load via qRT-PCR

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Viral load was quantified in capsid gene copy number units using the qRT-PCR method essentially as described previously [14 (link)]. Reactions were performed in duplicate using the SensiFAST SYBR No-ROX One-Step kit (Bioline, Alexandra, NSW, Australia) on a BioRad CFX96/C1000 thermal cycler platform using the primers RHDV-RT2_fw 5’-ACCCAGTACGGCACRGGCTCCCAACCAC-3′ and RHDV-RT2_rv 5’-CTATCTCCATGAAACCAGATGCAAAGGT-3′ [15 (link)].

Hall R.N., Capucci L., Matthaei M., Esposito S., Kerr P.J., Frese M, & Strive T. (2017). An in vivo system for directed experimental evolution of rabbit haemorrhagic disease virus. PLoS ONE, 12(3), e0173727.

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Quantifying Cytomegalovirus DNA in Plasma

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Peripheral blood samples were obtained from patients at admission before coronary angiography and from controls at the time of examination. Blood was centrifuged at 3,000 g for 15 minutes. We extracted viral DNA from plasma using the viral DNA spin protocol QIAamp UltraSense Virus kit. Detection of cytomegalovirus was performed by means of quantitative real-time PCR on a Bio-Rad thermal cycler CFX 96 C1000 using primer/probe sets for cytomegalovirus and human endogenous retrovirus 3 (ERV-3)^{29 (link)} (Table 2). Load of cytomegalovirus was expressed as copy number of cytomegaloviral DNA per 1 ml of plasma. The specificity of the PCR reaction was confirmed with purified cytomegalovirus as positive control and with human cord blood DNA as negative control. The threshold level of 1,000 copies of cell-free cytomegaloviral DNA per 1 ml of plasma was used as a categorical parameter of the presence of highly productive cytomegalovirus infection^{30 (link)}.

Lebedeva A., Maryukhnich E., Grivel J.C., Vasilieva E., Margolis L, & Shpektor A. (2019). Productive cytomegalovirus infection is associated with impaired endothelial function in ST-elevation myocardial infarction. The American journal of medicine, 133(1), 133-142.

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Detailed qRT-PCR Workflow for RLR Signaling

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The TRIzol reagent (Thermo Fisher Scientific) was used for RNA extraction. The concentration of total RNA was determined by using a spectrophotometer (NanoDrop 2000; Thermo). RNase-free DNase I (Thermo) was used to remove genomic DNA remnants at 37°C for 30 min. The cDNA was synthesized using the RevertAid first strand cDNA synthesis kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. qRT-PCR was performed on a Bio-Rad CFX96 C1000 thermal cycler using iQ SYBR green supermix (Bio-Rad, Singapore) under the following conditions: 3 min at 95°C, followed by 45 cycles of 10 s at 95°C, 15 s at 60°C, and 10 s at 72°C. All reactions were performed in triplicate and the mean value recorded. Those genes involved in the RLR antiviral signaling pathway (mda5, rig-I, mavs, tbk1, irf3, irf7, ifn1, and mx1) and the genes involved in cholesterol uptake and biosynthesis (lxra, ldlr, srebf1, fdft1, and hmgcr) were chosen. The housekeeping genes, including β-actin, EF-1α, and 18S rRNA were used for normalizing cDNA amounts. The fold changes relative to the control group were calculated using the threshold cycle (2^−ΔΔ^CT) method. All primers used for qRT-PCR are shown in Table S1 in the supplemental material.

Song Y.J., Zhang J., Xiao J., Feng H., Xu Z., Nie P, & Chang M.X. (2023). Piscine Vitamin D Receptors Vdra/Vdrb in the Absence of Vitamin D Are Utilized by Grass Carp Reovirus for Promoting Viral Replication. Microbiology Spectrum, 11(4), e01287-23.

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