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Cfx connect qpcr platform

Manufactured by Bio-Rad
Sourced in United States

The CFX Connect qPCR platform is a real-time PCR detection system designed for quantitative gene expression analysis. It features a compact design and supports a range of sample types and reaction volumes. The system provides precise temperature control and sensitive fluorescence detection to enable reliable and reproducible qPCR results.

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2 protocols using cfx connect qpcr platform

1

Quantitative RT-qPCR for Host and Viral Gene Expression

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Total RNA was extracted from cell lysates using the RNeasy Plus kit (Qiagen) according to the manufacturer’s protocol and reverse-transcribed [25 (link)] with iScriptTM Reverse Transcription Supermix (Biorad, Hercules, CA, USA) using random hexamer primers. The expression levels of target genes and M1 were measured using the CFX Connect qPCR platform (BioRad) and iTaqTM Universal SYBR® Green Supermix (BioRad). The qPCR thermal cycle was as follows: initial denaturation 3 min at 95°C, followed by 40 cycles including 10 s at 95°C and 30 s at 60°C. Each point was performed in triplicate. To make sure that the primers produced a single and specific PCR amplification product, a dissociation curve was carried out at the end of the PCR cycle. Host genes were quantified using the comparative ΔΔCt method. The mean ΔCt obtained in mock-infected cells for each gene was used as calibrator, after normalization to endogenous HPRT, GAPDH and β-tubulin housekeeping genes. Details of the primers are provided in supplemental materials. Chicken RNA samples were processed as previously described for host response [32 (link)] and viral RNA quantifying [37 (link)].
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2

Quantitative RT-PCR Analysis of Gene Expression

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Total RNA was extracted from OM primary culture cells using the Trizol method and subsequently treated with DNase I. OligodT first strand cDNA were synthesized from 5 μg total RNA by the Superscript II reverse transcriptase (Invitrogen, Cergy Pontoise, France) following the manufacturer recommendations. 5 µL of 200-fold diluted cDNA templates were added to a 15 μL reaction mixture containing 200 nM primers (sequences are shown in Additional Table 1) and SYBR Green GoTaq® qPCR Master Mix (Promega, Charbonnieres, France). The expression levels of target genes were measured using the CFX Connect qPCR platform (BioRad, Hercules, CA, USA). A dissociation curve was carried out at the end of the PCR cycle to verify efficiency of primers to produce a single and specific PCR amplification. Quantification was achieved using the ΔΔCt method, and mRNA expression was normalized to the expression level of β-actin (Francois et al., 2016). An efficiency corrective factor was applied for each primer pair.
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