This data set created a dilution series consisting of four 10-fold serial dilution points from 15,000 to 15 molecules, using 10 ng / μl yeast tRNA as a carrier (Roche) and created NTC samples of the same dilution. qPCR was done on a CFX 384 instrument (Bio-Rad). QPCR was performed on a CFX 384 instrument (Bio-Rad) using a 96-well pipetting robot (Tecan Freedom Evo 150). Amplification reactions were performed in 8 μl samples containing 0.4 μl forward and 0.4 μl reverse primer (5 μM each), 0.2 μl nuclease-free water, 4 μl iQ SYBR Green Supermix (Bio-Rad) and 3 μl of standard oligonucleotide. In 384-well plates (Hard-Shell 384-well microplate and Microseal B clear using an adhesive seal (Bio-Rad)), for each of the 4 dilution points, a total of 94 replicate reactions were distributed. In addition, the NTC reaction was repeated 8 times [28 (link)]. This dataset will be referred to as ‘94-replicates-4-dilutions set’. And 44 (4 × 11) amplification curves of the MYCN gene with a diluted concentration of 15, 150, 1500,15,000(11 replicated experiments for each group) were used for subsequent analysis.
Cfx384 instrument
Manufactured by Bio-Rad
Sourced in United States
The CFX384 instrument is a real-time PCR detection system designed for high-throughput nucleic acid analysis. It features a 384-well block format and can perform quantitative PCR (qPCR) and reverse transcription qPCR (RT-qPCR) experiments. The system is capable of temperature cycling and fluorescence detection to enable precise quantification of target nucleic acid sequences.
Automatically generated - may contain errors
Lab products found in correlation
Trizol reagent, by Thermo Fisher Scientific (11 mentions)
Primescript rt reagent kit, by Takara Bio (2 mentions)
Power sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Rneasy mini kit, by Qiagen (2 mentions)
Hard shell 384 well microplate, by Bio-Rad (2 mentions)
Microseal b clear, by Bio-Rad (2 mentions)
Yeast trna, by Roche (2 mentions)
Iq sybr green supermix, by Bio-Rad (2 mentions)
Freedom evo 150, by Tecan (2 mentions)
Iscript cdna synthesis kit, by Bio-Rad (2 mentions)
Iscript select cdna synthesis kit, by Bio-Rad (2 mentions)
Neb lunascript rt supermix kit, by New England Biolabs (2 mentions)
Taqman probe, by Bio-Rad (2 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (2 mentions)
Cfx maestro software, by Bio-Rad (2 mentions)
Sybr green supermix, by Bio-Rad (2 mentions)
Superscript 3 first strand synthesis system, by Thermo Fisher Scientific (2 mentions)
Sybr green master mix, by Bio-Rad (2 mentions)
Dnase 1, by Thermo Fisher Scientific (2 mentions)
Sybr green fast mixture, by Bio-Rad (1 mentions)
32 protocols using cfx384 instrument
1
Dilution Series for qPCR Optimization
2
Dilution Series for qPCR Optimization
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This data set created a dilution series consisting of four 10-fold serial dilution points from 15,000 to 15 molecules, using 10 ng / µl yeast tRNA as a carrier (Roche)
and created NTC samples of the same dilution. qPCR was done on a CFX 384 instrument (Bio-Rad). QPCR was performed on a CFX 384 instrument (Bio-Rad) using a 96-well pipetting robot (Tecan Freedom Evo 150). Amplification reactions were performed in 8µl samples containing) 0.4µl forward and 0.4µl reverse primer (5µM each), 0.2µl nuclease-free water, 4µl iQ SYBR Green Supermix (Bio-Rad) and 3µl of standard oligonucleotide. In 384-well plates (Hard-Shell 384-well microplate and Microseal B clear using an adhesive seal (Bio-Rad)), for each of the 4 dilution points, a total of 94 replicate reactions were distributed. In addition, the NTC reaction was repeated 8 times [9] . This dataset will be referred to as '94replicates-4-dilutions set'. Since our system has 96 reaction channels, we select 44 curve data with concentration gradient from data set for analysis.
and created NTC samples of the same dilution. qPCR was done on a CFX 384 instrument (Bio-Rad). QPCR was performed on a CFX 384 instrument (Bio-Rad) using a 96-well pipetting robot (Tecan Freedom Evo 150). Amplification reactions were performed in 8µl samples containing) 0.4µl forward and 0.4µl reverse primer (5µM each), 0.2µl nuclease-free water, 4µl iQ SYBR Green Supermix (Bio-Rad) and 3µl of standard oligonucleotide. In 384-well plates (Hard-Shell 384-well microplate and Microseal B clear using an adhesive seal (Bio-Rad)), for each of the 4 dilution points, a total of 94 replicate reactions were distributed. In addition, the NTC reaction was repeated 8 times [9] . This dataset will be referred to as '94replicates-4-dilutions set'. Since our system has 96 reaction channels, we select 44 curve data with concentration gradient from data set for analysis.
3
Profiling miR-125b-1-3p and Candidate Genes
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The expression levels of miR-125b-1-3p were analyzed in human peripheral blood (clinical cohorts), in the hippocampi of animals (adult male rats exposed or not to PNS), as well as in HPC0A07/03C cells (treated or not with cortisol 100µM) by Real Time PCR using the CFX384 instrument (Bio-Rad Laboratories, Hercules, CA, USA) and the TaqMan MicroRNA Assays (Thermofisher, Waltham, MA, USA), following the manufacturer's instructions. The relative expression of miR-125b-1-3p was normalized to the levels of three housekeeping genes RNU44, RNU48 and RNU49 in humans, and to the levels of U6 and U87 in rats. All the reactions were performed in triplicates.
The expression levels of the candidate genes Signal Transducer And Activator Of Transcription 1 (STAT1), Forkhead Box O1 (FoxO1), Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1), and of the housekeeping genes Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and Actin Beta (ACTB) were evaluated by using TaqMan Assays (Thermofisher, Waltham, MA, USA) on the CFX384 instrument (Bio-Rad Laboratories, Hercules, CA, USA), following the manufacturer's instructions.
The Pfaffl Method was used to determine the relative expression values of miR-125b-1-3p and of genes of interest (Pfaffl, 2001) .
The expression levels of the candidate genes Signal Transducer And Activator Of Transcription 1 (STAT1), Forkhead Box O1 (FoxO1), Nuclear Receptor Subfamily 3 Group C Member 1 (NR3C1), and of the housekeeping genes Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) and Actin Beta (ACTB) were evaluated by using TaqMan Assays (Thermofisher, Waltham, MA, USA) on the CFX384 instrument (Bio-Rad Laboratories, Hercules, CA, USA), following the manufacturer's instructions.
The Pfaffl Method was used to determine the relative expression values of miR-125b-1-3p and of genes of interest (Pfaffl, 2001) .
4
Quantitative PCR and Cytokine Analysis
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At the indicated time points per experiment, total RNA was extracted using TRIzol reagent (Thermo Fisher) and reverse-transcribed using the PrimeScript RT Reagent kit (Takara) according to the manufacturer’s instructions. The CFX384 instrument (Bio-Rad) with SYBR green fast mixture (Bio-Rad) was used for quantitative real-time PCR analysis. Expression levels were quantified using the 2−ΔΔCt method and normalized to GAPDH. The primer sequences are listed in Supplementary Table 1
5
Quantitative RT-PCR Analysis of Megakaryocytic Differentiation
6
Gene Expression Analysis of Murine Intervertebral Discs
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Intact thoracic IVDs (inclusive of NP, AF, and CEP) (4–5 per mouse; 5–8 mice per diet/per timepoint) were isolated by microdissection, placed in TRIzol reagent (Thermo Fisher Canada: Mississauga, ON, CAN) and homogenized using a PRO250 tissue homogenizer (PRO Scientific: Oxford, CT, USA). RNA was extracted according to manufacturer’s instructions, quantified using a NanoDrop 2000 spectrophotometer (Thermo Fisher Canada: Mississauga, ON, CAN), and 0.5 μg was reverse transcribed into complementary DNA (cDNA) (iScript; Bio-Rad Laboratories (Canada): Mississauga, ON, CAN). Gene expression was assessed by real-time PCR using a Bio-Rad CFX384 instrument. PCR analyses were run in triplicate using 120 ng of cDNA per reaction and 310 nM forward and reverse primers with 2x SsoFast EvaGreen Supermix (Bio-Rad Laboratories (Canada): Mississauga, ON, CAN) using optimized PCR parameters and primers (Supplementary Table 2 ). Primers were designed and validated to have efficiency values between 90 and 120%. Transcript levels were calculated relative to a 6-point standard curve made from pooled cDNA generated from murine IVD explants treated with lipopolysaccharide for 4 days (50 mg/mL; Thermo Fisher Canada: Mississauga, ON, CAN).
7
Quantitative Analysis of miRNA Expression
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Reverse transcription was performed using miScript II RT Kit (Qiagen, cat# 218161) using 500 ng of total RNA. Samples were incubated at 37 °C for 1 h followed by an inactivation step at 95 °C for 5 min.
Quantitative PCR analysis was performed using miScript SYBR Green PCR Kit (Qiagen, cat#: 218073). Primers for mmu-miR-98-5p, mmu-let-7d-3p, mmu-let-7 g-5p, Hs_RNU6-2_11, SNORD68, mmu-miR-140-3p, mmu-miR-150-5p, mmu-miR-92a-3p, mmu-miR-148a-3p, mmu-miR-214-3p and mmu-miR-340-5p were purchased from Qiagen. Quantitative PCR was performed in triplicates from three or four biological replicates using cDNA corresponding to 10 ng of template total RNA. PCR was performed using CFX384 ™ instrument (Bio-Rad) with the cycling conditions 95 °C for 15 min, 94 °C for 15 s, 55 °C for 30 s and 70 °C for 30 s; the last three steps were repeated for 40 cycles.
Quantitative PCR analysis was performed using miScript SYBR Green PCR Kit (Qiagen, cat#: 218073). Primers for mmu-miR-98-5p, mmu-let-7d-3p, mmu-let-7 g-5p, Hs_RNU6-2_11, SNORD68, mmu-miR-140-3p, mmu-miR-150-5p, mmu-miR-92a-3p, mmu-miR-148a-3p, mmu-miR-214-3p and mmu-miR-340-5p were purchased from Qiagen. Quantitative PCR was performed in triplicates from three or four biological replicates using cDNA corresponding to 10 ng of template total RNA. PCR was performed using CFX384 ™ instrument (Bio-Rad) with the cycling conditions 95 °C for 15 min, 94 °C for 15 s, 55 °C for 30 s and 70 °C for 30 s; the last three steps were repeated for 40 cycles.
8
Profiling Chondrogenic and Osteogenic Markers
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Expression profiles of Sox5, Col2a1, Acan, Runx2, Sp7 and Bmp2 in callus samples were validated as markers for progression of chondrogenesis and osteogenesis by qRT-rtPCR (Jeyakumar et al., 2017 (link); Jensen et al., 2010 (link)). Reverse transcription was performed using SensiFAST ™ cDNA Synthesis kit (Bioline) with 500 ng of total RNA. The qPCR step reaction was performed in triplicates using DyNAmo Flash SYBR Green qPCR kit (ThermoFisher Scientific, cat#F-415L) and 10 ng of total RNA as a template with primers listed in Supplemental File 1 : STable 1. PCR was performed using CFX384 ™ instrument (Bio-Rad) with the cycling conditions 95 °C for 15 min, 95 °C for 15 s, 60 °C for 30 s and 72 °C for 30 s. The last three steps were repeated for 40 cycles. ΔCT was calculated using the geometric mean of Tubb_5 and Actb gene expression.
9
Gene Expression Profiling in Infected Cells
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In infected LoVo or JEG-3 cells, RNA was extracted at, respectively, 18 or 20 h p.i. with an RNeasy minikit (Qiagen) as recommended by the manufacturer, using 1 column per well of a 24-well plate and biological duplicates for each condition. Genomic DNA was removed by treatment with a Turbo DNA-free kit (Ambion). cDNAs were generated from 1 to 2 µg total RNA using the RT2 HT first-strand kit (Qiagen/SABiosciences).
qPCR was performed on a CFX384 instrument (Bio-Rad) using Kapa SYBR Fast universal master mix (Kapa Biosystems) as specified by the supplier. Each reaction was performed in triplicate. RT-qPCR primers for YWHAZ, IFI44L, IFITM1, and IL6 were predesigned, validated RT2 qPCR primer pairs from Qiagen/SABiosciences. Other primer pairs were selected from the literature, from primer databases (RTPrimerDB,http://medgen.ugent.be/rtprimerdb/ , or qPrimerDepot, http://primerdepot.nci.nih.gov/ ) and validated for efficiency. Data were analyzed by the ∆∆CT method. Target gene expression data were normalized to the relative expression of the YWHAZ reference gene. The displayed results are representative of three independent experiments in LoVo cells and two experiments in JEG-3 cells.
qPCR was performed on a CFX384 instrument (Bio-Rad) using Kapa SYBR Fast universal master mix (Kapa Biosystems) as specified by the supplier. Each reaction was performed in triplicate. RT-qPCR primers for YWHAZ, IFI44L, IFITM1, and IL6 were predesigned, validated RT2 qPCR primer pairs from Qiagen/SABiosciences. Other primer pairs were selected from the literature, from primer databases (RTPrimerDB,
10
Quantifying Intestinal Inflammation and Gene Expression
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Intestinal inflammation was assessed based on measures of fecal calprotectin (30-CALPHU-CH01) and myeloperoxidase (30-6630; Alpco). Assays were performed in accordance with the manufacturer’s instructions, and an easy stool extraction device (30-EZEX-100) was used to accurately measure stool mass prior to sample analysis. Intra- and inter-assay CVs of both proteins were <3.0% and <7.3%, respectively.
Whole blood pro-inflammatory gene expression was measured by real-time quantitative polymerase chain reaction. Total RNA was extracted using a RiboPure-Blood Kit (ThermoFisher Scientific, AM 1928). RNA yield and purity were assessed using a BioSpec-nano spectrophotometer (Shimadzu, Tokyo, Japan), and integrity was determined following native agarose gel (2%) electrophoresis. cDNA was synthesized from high-quality RNA using an iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). Primers (IL-8, MCP1, TNFα, IL6, TLR4, p65, β-actin) were purchased from Integrated DNA Technologies (Table S3 ) and validated from in-house melt curve and standard curve analysis. Analysis was performed using a Bio-Rad CFX384 instrument with SYBR Green Supermix (Bio-Rad). Target gene expression, with normalization to β-actin, was calculated using the 2−ΔΔCT method [38 (link)].
Whole blood pro-inflammatory gene expression was measured by real-time quantitative polymerase chain reaction. Total RNA was extracted using a RiboPure-Blood Kit (ThermoFisher Scientific, AM 1928). RNA yield and purity were assessed using a BioSpec-nano spectrophotometer (Shimadzu, Tokyo, Japan), and integrity was determined following native agarose gel (2%) electrophoresis. cDNA was synthesized from high-quality RNA using an iScript cDNA synthesis kit (BioRad, Hercules, CA, USA). Primers (IL-8, MCP1, TNFα, IL6, TLR4, p65, β-actin) were purchased from Integrated DNA Technologies (
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