After two days of co-culture, the flow was stopped, and the GMPS set-up was removed from the incubator. Via the seed port, PBS++ was used to wash the apical channel, and 300 μL RNA lysis buffer (cat no. T2010, New England Biolabs Inc.) was applied to the channel and vigorously pipetted. RNA was purified following the manufacturer’s protocol (cat no. T2010, New England Biolabs Inc.). Oligo(dT)12–18 (cat no. 18418012, Thermo Fisher) was used to reverse transcribe Caco-2 RNA (C1000 Touch Thermal Cycler, BioRad). The resulting cDNA was cleaned and concentrated (cat no. D40144, Zymo Research) and 1 μL was used for qPCR. An SYBR Green-based qPCR master mix (cat no. M3003, New England Biolabs Inc.) and primers (Table S3 .) were used to amplify genes of interest (C1000 Thermal Cycler with CFX96 Real-Time System, BioRad). Primers were made with NCBI Primer Blast.75 The target gene, GADD45a, was normalized to GAPDH. Relative expression differences were calculated using the delta-delta Ct method.
Cfx96 real time system with c1000 thermal cycler
Manufactured by Bio-Rad
The CFX96 Real-Time System with C1000 Thermal Cycler is a laboratory instrument designed for real-time PCR (polymerase chain reaction) analysis. It combines a thermal cycler and a fluorescence detection system to enable quantitative, real-time monitoring of nucleic acid amplification. The instrument provides precise temperature control and accurate data collection for a wide range of real-time PCR applications.
Automatically generated - may contain errors
Lab products found in correlation
Sybr green, by Thermo Fisher Scientific (4 mentions)
Trizol reagent, by Thermo Fisher Scientific (3 mentions)
Specific oligonucleotides, by Thermo Fisher Scientific (3 mentions)
High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (3 mentions)
Purelink rna mini kit, by Thermo Fisher Scientific (3 mentions)
Oligo dt 12 18, by Thermo Fisher Scientific (2 mentions)
C1000 touch thermal cycler, by Bio-Rad (2 mentions)
Rna lysis buffer, by New England Biolabs (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions)
Agilent rna 6000 kit, by Agilent Technologies (2 mentions)
Ssoadvanced universal sybr green supermix, by Bio-Rad (1 mentions)
Cfx manager 3, by Bio-Rad (1 mentions)
Tempase hot start dna polymerase, by Avantor (1 mentions)
Taqman primer probe, by Thermo Fisher Scientific (1 mentions)
Superscript first strand synthesis system for rt pcr, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Quantitect reverse transcription kit, by Qiagen (1 mentions)
13 protocols using cfx96 real time system with c1000 thermal cycler
1
Quantifying GADD45a Expression in Caco-2 Cells
2
Quantitative Transcript Analysis of Bacteria
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Bacteria were grown overnight at 20°C in KB broth containing 50 μg/mL rifampicin to an OD600 = ~1.0. Cells were centrifuged for 10 min at 21,000 x g and the resulting bacterial pellets were frozen in liquid nitrogen and stored at -80°C prior to use. RNA was extracted from the bacterial pellets and 500 ng converted into cDNA in 25 μL reactions as described previously [9 (link)]. Quantitative RT-PCR (qRT-PCR) reactions were performed in 10 μL volumes using 5 μL of SsoAdvanced Universal SYBR Green Supermix (Bio-Rad), 4 μL of a mix containing 0.5 μM of each transcript-specific primer (S2 Table ), and 1 μL of cDNA. SYBR Green fluorescence from each qRT-PCR reaction was monitored using the C1000 Thermal Cycler with CFX96 Real-Time System (Bio-Rad). Relative abundance of transcripts was calculated relative to gyrA using the formula Transcript Abundance = PCR efficiency-(Ct[gene]-Ct[gyrA]) [48 (link)]. PCR efficiency values were calculated for each qRT-PCR reaction using LinRegPCR [49 ].
3
Caco-2 Cells RNA Isolation and qPCR
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After two days of co-culture, the flow was stopped, and the GMPS set-up was removed from the incubator. Via the seed port, PBS++ was used to wash the apical channel, and 300 μL RNA lysis buffer (cat no. T2010, New England Biolabs Inc.) was applied to the channel and vigorously pipetted. RNA was purified following the manufacturer’s protocol (cat no. T2010, New England Biolabs Inc.). Oligo(dT)12–18 (cat no. 18418012, Thermo Fisher) was used to reverse transcribe Caco-2 RNA (C1000 Touch Thermal Cycler, BioRad). The resulting cDNA was cleaned and concentrated (cat no. D40144, Zymo Research) and 1 μL was used for qPCR. A SYBR Green-based qPCR master mix (cat no. M3003, New England Biolabs Inc.) and primers (Table S3 .) were used to amplify genes of interest (C1000 Thermal Cycler with CFX96 Real-Time System, BioRad). Primers were made with NCBI Primer Blast76 . The target gene, GADD45a, was normalized to GAPDH. Relative expression differences were calculated using the delta-delta Ct method.
4
qPCR Genotyping Assay Protocol
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The qPCR assays with dual-labeled probes of Bouuaert et al. (2021) were used for genotyping54 (link). Briefly, for each gDNA sample, genotyping assays were performed in a total volume of 10 µL with 1 × KEY buffer, 250 nM of each primer, 250 nM of each dual-labeled probe, 200 µM of each dNTP, 0.5 U TEMPase Hot Start DNA Polymerase (VWR) and 20 ng gDNA. Primer and probe sequences can be found in54 (link). The Bio-Rad C1000™ Thermal Cycler with CFX96™ Real-Time System was set at one cycle of 95 °C for 14′40″, followed by 60 cycles of [95 °C for 20″ followed by 40″ of the assay-specific annealing/elongation/signal detection temperature]54 (link). Data analysis and allelic discrimination plot construction was done with the Bio-Rad CFX Manager 3.1 Software.
5
Quantifying Gene Expression in Infected Lungs
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Total RNA from infected lungs or cultured macrophages was extracted with TRIzol Reagent (Invitrogen), according to the manufacturer’s instructions. cDNA was synthesized using the SuperScript First-Strand Synthesis System for RT-PCR (Thermo Scientific). Target gene mRNA expression was quantified by real-time PCR (Bio-Rad CFX96 Real-Time System with C1000 Thermal Cycler) and normalized to Hprt1 or Ubiquitin mRNA levels. Target gene mRNA expression was quantified using SYBR Green (Thermo Scientific) and specific oligonucleotides (Invitrogen) for Tnf ([F] 5′-GCC ACC ACG TCT TCT GTC T-3′, [R] 5′-TGA GGG TCT GGG CCA TAG AAC-3′) and Ubiquitin ([F] 5′-TGG CTA TTA ATT ATT CGG TCT GCAT-3′, [R] 5′-GCA AGT GGC TAG AGT GCA GAG TAA-3′) or TaqMan primer probes (Applied Biosystems) for Nos2 (Mm00440502_m1), Arg1 (Mm00475988_m1), Ym1 (Mm00657889_mH), Fizz1 (Mm00445109_m1), Il4 (Mm00445260_m1), Il5 (Mm00439646_m1), Il13 (Mm00434204_m1), Il10 (Mm00439614_m1) and Hprt1 (Mm00446968_m1).
6
Quantifying PCCA and PCCB mRNA Levels
7
Quantitative RT-PCR Profiling of Lung Transcripts
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Quantitative RT-PCR (qRT-PCR) was performed as previously described (66 (link)). Total RNA from whole lungs was extracted using TRIzol (Invitrogen) from which cDNA was generated using the GRS cDNA Synthesis Mastermix (Grisp), following the manufacturer’s instructions. The resultant cDNA template was used to quantify the expression of target genes by qRT-PCR (CFX96 Real-Time System with C1000 Thermal Cycler, Bio-Rad) and normalized to ubiquitin mRNA levels using the ΔCt method. Target gene mRNA expression was quantified using SYBR Green (Thermo Fisher Scientific) and specific oligonucleotides (Invitrogen).
8
Hepatocyte RNA Isolation and RT-qPCR Analysis
9
Quantification of Gene Expression in Infected Lungs
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Total RNA from infected lungs was extracted using TRIzol reagent (Invitrogen) following the manufacturer’s instructions. cDNA was generated from 1 μg of total RNA using the GRS cDNA Synthesis Master Mix (GRiSP, Porto, Portugal) following the manufacturer’s instructions. The resultant cDNA template was used to quantify the expression of target genes by real-time (RT)-PCR (Bio‐Rad CFX96 Real‐Time System with C1000 Thermal Cycler) using the following protocol: one cycle of 95°C for 3 min, followed by 40 cycles of a two-stage temperature profile of 95°C for 3 s and 60°C for 30 s. Gene expression was normalized to ubiquitin mRNA levels using the ΔCt method. Target gene mRNA expression was quantified using SYBR Green (Thermo Scientific, Waltham, MA, USA) and specific oligonucleotides (Table 1
10
Real-Time PCR Gene Expression Analysis
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Total RNA was extracted using Triple XTractor (GB023.0100, Grisp, Porto, Portugal) according to the manufacturer’s instructions. cDNA was generated from 1 μg of RNA using the GRS cDNA Synthesis Master Mix (GK81.0100, Grisp) following the manufacturer’s instructions. The resultant cDNA template was used to quantify the expression of target genes by real-time PCR (Bio-Rad CFX96 Real-Time System with C1000 Thermal Cycler), and normalized to Ubiquitin mRNA levels using the ΔCt method (1.8^(Housekeeping gene mRNA expression—Target gene mRNA expression) × 100,000). Target gene mRNA expression was quantified using SYBR Green (Thermo Fisher Scientific, Waltham, MA, USA) and specific oligonucleotides (Invitrogen).
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