Cfx96 384 instruments

Manufactured by Bio-Rad

The CFX96/384 instruments are real-time PCR detection systems designed for quantitative gene expression analysis and genotyping. The systems provide thermal cycling, fluorescence detection, and data analysis capabilities to support a range of real-time PCR applications.

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Lab products found in correlation

Gotaq qpcr master mix kit, by Promega (4 mentions) Cfx manager software v3, by Bio-Rad (4 mentions) Reliaprep rna cell miniprep system, by Promega (3 mentions) Cdna synthesis kit, by Meridian Bioscience (2 mentions) Fastprep 24 5g, by MP Biomedicals (1 mentions) Dna engine opticon 2, by Bio-Rad (1 mentions) Absolute rna microprep kit, by Agilent Technologies (1 mentions) Prism software v4, by GraphPad (1 mentions) Gotaq 1 step rt qpcr, by Promega (1 mentions) Tetro cdna synthesis kit, by Meridian Bioscience (1 mentions) Sybr green master mix, by Promega (1 mentions)

Close spelling variants of this product

CFX96/384 fluorescent quantitative PCR instrument CFX96 instrument CFX96/384 CFX96

4 protocols using cfx96 384 instruments

RNA Extraction and RT-qPCR Analysis

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Mouse tissues were homogenized in 500 ul of RNA lysing buffer in homogenizer (MP Biomedicals, FastPrep 24 5G) using metal beads (MP Biomedicals, 116925100) 2 x 60s cycle and then centrifuged at 13 000 rpm for 10 min. at 4°C. Supernatant was taken to a fresh tube and RNA was isolated using ReliaPrep™ RNA Cell Miniprep System (Promega). 250 ng to 1 ug RNA was subjected to first strand cDNA synthesis using Bioline kit. RT-qPCR analysis was carried out using CFX96/384 instruments (Bio-Rad) and the GoTaq qPCR Mastermix Kit (Promega). RT-qPCR primer sequences are listed in Supplementary
Table 1. Expression analysis used CFX Manager Software v3.1 (Bio-Rad), with samples normalized to a combination of TATA box-binding protein and glyceraldehyde 3-phosphate dehydrogenase expression.

Mularczyk E.J., Singh M., Godwin A.R., Galli F., Humphreys N., Adamson A.D., Mironov A., Cain S.A., Sengle G., Boot-Handford R.P., Cossu G., Kielty C.M, & Baldock C. (2018). ADAMTS10-mediated tissue disruption in Weill–Marchesani syndrome. Human Molecular Genetics, 27(21), 3675-3687.

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Quantitative RT-PCR Analysis of Gene Expression

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RNA was isolated from cells using ReliaPrep™ RNA Cell Miniprep System (Promega). Up to 2 μg RNA was used to generate cDNA, using a cDNA synthesis kit (Bioline). RT-qPCR analysis was carried out using CFX96/384 instruments (Bio-Rad) and the GoTaq qPCR Mastermix Kit (Promega). RT-qPCR primer sequences are listed in Supplementary Table 1 and were purchased from Eurofins. Due to very low expression levels, ADAMTS10 gene expression analysis was performed using TaqMan probes (Hs01548644_g1 (ADAMTS10), ThermoFisher Scientific), with matching reference gene primers. Expression analysis used CFX Manager Software v3.1 (Bio-Rad), with samples normalized to a combination of TATA box binding protein (TBP) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Gene expression data are in Supplemental Information.

Cain S.A., Mularczyk E.J., Singh M., Massam-Wu T, & Kielty C.M. (2016). ADAMTS-10 and -6 differentially regulate cell-cell junctions and focal adhesions. Scientific Reports, 6, 35956.

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Quantitative Real-Time PCR Analysis

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RNA was isolated from ARPE-19, HDF cells and podocytes using an Absolute RNA Microprep Kit (Agilent Technologies). 500 ng RNA was used to generate cDNA using a cDNA synthesis kit (Bioline). Real-time qPCR analysis was carried out using either DNA Engine Opticon 2 (MJ Research Inc.) or CFX96/384 instruments (Bio-Rad) and the GoTaq qPCR Mastermix Kit (Promega). Expression analysis was performed in triplicate using CFX Manager software v3.0 (Bio-Rad), with samples normalised to a combination of TATA box binding protein (TBP) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression unless otherwise stated. Gene expression data (relative to control cell expression) from across replicate experiments was either entered directly into Prism software v4.03 (GraphPad Software Inc.) or (when stated in figure legends) first imported into the ‘Gene Study’ functionality of CFX Manager prior to transfer of expression, s.e.m., and n-values into Prism. Prism software was used for analysis via two-way ANOVA with Bonferroni post-tests. The oligonucleotide primers used for all qPCR reactions are shown in supplementary material Table S1.

Baldwin A.K., Cain S.A., Lennon R., Godwin A., Merry C.L, & Kielty C.M. (2014). Epithelial–mesenchymal status influences how cells deposit fibrillin microfibrils. Journal of Cell Science, 127(1), 158-171.

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Quantifying G2L1 Expression in U2OS Cells

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Total RNA was extracted from U2OS cells using the ReliaPrep RNA Cell Miniprep System (Promega, UK), 48 hours post-transfection. 0.5 μg total RNA was used for cDNA synthesis with MMLV reverse transcriptase and either random hexamers or oligo(dT) primers together with control RNA template according to manufacturer manual (Tetro cDNA Synthesis Kit, Bioline, UK). RT-qPCR was performed using SYBR Green Mastermix (GoTaq 1-Step RT-qPCR, Promega, UK). Primers for G2L1 were designed using the NCBI primer-BLAST oligos designer web tool.
Primer sequences are:
Forward primer: CTC ATC TTT GTG CGG GTG CT
Reverse primer: AGG TAA TGC TCC AGC GTG TC
The efficiency of each primer set for RT-qPCR was determined to be between 95 and 100%. Primers were purchased from Life Technologies (UK). Real-time qPCR analysis was carried out using CFX96/384 instruments (Bio-Rad) and the GoTaq qPCR Mastermix Kit (Promega). Expression analysis was performed in triplicate using CFX Manager software v3.0 (Bio-Rad), with samples normalised to a combination of TATA box binding protein (TBP) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression. Gene expression data (relative to control cell expression) from across replicate experiments was entered directly into Microsoft Excel, where it was used for analysis using One-way ANOVA.

Nazgiewicz A., Atherton P, & Ballestrem C. (2019). GAS2-like 1 coordinates cell division through its association with end-binding proteins. Scientific Reports, 9, 5805.

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