Total RNA from cells was isolated, as previously described [19 (link)]. Primers for the amplification of murine MMP-13, MMP-14, and S26 were described elsewhere [19 (link)]. For quantitative real-time PCR analysis, reversed transcribed RNA was used for real-time PCR using the StepOne RealTime (Applied Biosystems, Thermo Scientific, Bonn, Germany). Five microliters of sample cDNA were added to the master mix, and the amplification performed in a total volume of 20 µL for 40 cycles. The thermal cycling conditions were set to 50 °C for 2 min and 95 °C for 10 min, followed by 40 cycles of amplification at 95 °C for 15 s and 60 °C for 1 min for each cycle. Primers used for real-time amplification were as follows: decorin sense 5′-TGAGCTTCAACAGCATCACC-3′, antisense 5′-AAGTCATTTTGCCCAACTGC-3′; collagen type I sense 5′-CCCCTGGTCTTACTGGGAAC-3′, antisense 5′-AGCAGGTCCTTGGAAACCTT-3′.
Stepone realtime
Manufactured by Thermo Fisher Scientific
Sourced in United States
The StepOne RealTime is a real-time PCR system designed for gene expression analysis and quantitative PCR applications. It provides accurate and reproducible results through its advanced optical system and temperature control technology.
Automatically generated - may contain errors
Lab products found in correlation
Nextera dna sample prep kit, by Illumina (1 mentions)
Kapa sybr fast qpcr, by Roche (1 mentions)
Cbot cluster generation system, by Illumina (1 mentions)
Agilent bioanalyzer 2100 system, by Agilent Technologies (1 mentions)
Superscript 2 rt kit, by Thermo Fisher Scientific (1 mentions)
Epicentre master pure rna purification kit, by Illumina (1 mentions)
Faststart universal sybr green master mix, by Roche (1 mentions)
Rq1 rnase free dnase, by Promega (1 mentions)
Cd117 conjugated magnetic beads, by Miltenyi Biotec (1 mentions)
Facschorus, by BD (1 mentions)
Facsmelody, by BD (1 mentions)
Stepone, by Thermo Fisher Scientific (1 mentions)
Taqman snp genotyping assay mix, by Thermo Fisher Scientific (1 mentions)
Taqman universal pcr master mix, by Thermo Fisher Scientific (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions)
5 protocols using stepone realtime
1
Quantitative Real-Time PCR Analysis of Murine Genes
2
Nextera Library Preparation and Sequencing
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The genomic library (nuclear and mitochondrial DNA) of N. amazoniensis was prepared with the Nextera DNA Sample Prep kit (Illumina, San Diego, CA, USA), using approximately 50 ng of DNA. The quality assessment of the genomic library was performed using the Agilent 2100 Bioanalyzer system (Agilent Technologies, Santa Clara, CA, USA) and the DNA sample was prepared with initial concentration of 10 nM. An aliquot of this library containing 2 nM of DNA fragments was quantified through an absolute quantification curve using KAPA SYBR® FAST qPCR (Kapa Biosystems, Wilmington, MA, USA) on a StepOne Real Time (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s recommendations.
The fragment indexing process was performed according to the manufacturer’s recommendations, using the HiSeq cBot Manifold (Illumina, San Diego, CA, USA) and the TrueSeq PE Cluster Kit v3—cBot HS reagents (Illumina, San Diego, CA, USA). Sixteen picomoles of the N. amazoniensis library were applied to the surface of the sequencing slide using the cBot Cluster Generation System (Illumina, San Diego, CA, USA) automated system.
The fragment indexing process was performed according to the manufacturer’s recommendations, using the HiSeq cBot Manifold (Illumina, San Diego, CA, USA) and the TrueSeq PE Cluster Kit v3—cBot HS reagents (Illumina, San Diego, CA, USA). Sixteen picomoles of the N. amazoniensis library were applied to the surface of the sequencing slide using the cBot Cluster Generation System (Illumina, San Diego, CA, USA) automated system.
3
Quantifying Cytokine Expression in Liver
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Total RNA was isolated from liver samples using Epicentre Master Pure RNA Purification kit (Illumina, San Diego, CA, USA), according to the instructions of the manufacturer. A DNAse (RQ1 RNAse-free DNAse, Promega, Madison, WI, USA) treatment in all RNA samples was performed. The cDNA was reversed transcribed from 2 μg of RNA, using random 6-mer oligonucleotides (5 ng/μl) and Superscript II RT kit (Invitrogen, Waltham, MA, USA).
The design and validation of TNF-α, IL-23, IFN-γ, IL-1β, IL-6, IL-8, IL-17A, IL-21, IL-10 and TGF-β specific primers are described inSupplementary Table S2
The design and validation of TNF-α, IL-23, IFN-γ, IL-1β, IL-6, IL-8, IL-17A, IL-21, IL-10 and TGF-β specific primers are described in
4
Quantification of Ext1 Gene Expression
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Peritoneal cavity single-cell suspensions were enriched with CD117 conjugated magnetic beads (Miltenyi) and sorted on BD FACSMelody (BD Biosciences) cell sorter running BD FACSChorus (BD Biosciences) software version 1.3.3 after labeling with antibodies described in the flow cytometry section. Sorted cells were incubated with proteinase K (Arrowtec) at 60°C for 5 min to lyse the cells, followed by the extraction of the genomic DNA using the gSYNC extraction kit (Geneaid) following the manufacturer’s instructions. Ext1 and Tfrc genes were amplified together using TaqMan Copy Number Assay ID Mm00613274_cn labeled with FAM and TaqMan Copy Number Reference Assay (cat. # 4458366 labeled with VIC), respectively (both from ThermoFisher) in a PCR thermal cycler (StepOne Real-Time, Applied Biosystems) with the following conditions: 1 cycle of denaturation/enzyme activation at 95°C for 10 min, 40 cycles of denaturation at 95°C for 15 sec followed by annealing/extension at 60°C for 1 min. Ext1 and Tfrc genes expression were analyzed on StepOne (Applied Biosystems) software version 2.3, and fold increase was calculated using the comparative CT (ΔΔCT) method on Microsoft Excel (Microsoft). Ext1 expression in each sample was normalized using the Tfrc gene expression.
5
DNA Extraction and Genotyping of MIR17HG
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Genomic DNA from nucleated WBCs of whole blood was extracted utilizing the Mini Kit of QIAamp DNA extraction designated for blood samples (Catalog #51104; Qiagen, Hilden, Germany). The NanoDrop ND‐1000 spectrophotometer (NanoDrop Technologies, Inc, Wilmington, DE) was utilized for estimation of the quantity and purity of genomic DNA. The intronic variant (rs4284505; A>G) of MIR17HG was characterized and genotyped by the TaqMan allelic discrimination polymerase chain reaction (PCR) using StepOne Real‐Time (Applied Biosystems). The protocol used was managed blindly with a final volume of 20 μL containing 20 ng of DNA template, 1 μL of TaqMan SNP Genotyping Assay Mix (assays ID: C__26557482_10; Applied Biosystems, Waltham), and 10 μL of TaqMan Universal PCR Master Mix. Proper negative controls were applied in each run.25 About 10% of the samples were randomly selected for assay replication, and the results yield 100% concordance.
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