Gene Expression Profiling via qRT-PCR

Total RNA was extracted using RNeasy Mini Kit (Qiagen) or Tri Reagent (Sigma Aldrich) according to the manufacturer's instructions. Contaminating DNA was removed by DNA-free Kit DNA Removal Kit (Ambion). RNA was reverse transcribed using High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). For Real time PCR, gene-specific primers for iNOS (20 (link)), Arg I (21 (link)), TNF-α (22 (link)), IL-10 (23 (link)), TGF-ß (24 (link)), MMP-2 (25 (link)), MMP-9 (26 (link)), Collagen-1, Collagen-3 (27 (link)), IL-6, 18S RNA (19 (link)), Fizz1/RELMα, Ym-1 (28 (link)), SPHK1, LIGHT (29 (link)), MertK (30 (link)), were used. All samples were run in triplicates. Relative gene expression levels were determined using PowerUP SYBR Green PCR Master Mix (Applied Biosystems) according to the manufacturer's recommended protocol with the following thermal cycling conditions: 2 min 50°C, 2 min 95°C, 40 cycles of 1 s 95°C and 30 s 60°C, and 4°C hold (QuantStudio 3 Real-Time PCR System, Applied Biosystems). Expression of target genes was normalized to the endogenous control 18S RNA gene. Fold expression was calculated using the 2^−ΔΔCt methods. In some experiments, the 2^ΔCt method was used to determine relative gene expression.

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Eghbalzadeh K., Georgi L., Louis T., Zhao H., Keser U., Weber C., Mollenhauer M., Conforti A., Wahlers T, & Paunel-Görgülü A. (2019). Compromised Anti-inflammatory Action of Neutrophil Extracellular Traps in PAD4-Deficient Mice Contributes to Aggravated Acute Inflammation After Myocardial Infarction. Frontiers in Immunology, 10, 2313.

Publication 2019

RNeasy Mini Kit Tri Reagent DNA-free Kit DNA Removal Kit High Capacity cDNA Reverse Transcription Kit PowerUP SYBR Green PCR Master Mix QuantStudio 3 Real-Time PCR System

18s rna Cdna Collagen Collagen 1 Gene Gene control Gene expression Il 10 Inos Light Mertk Mmp 2 Mmp 9 Primers Real time pcr Reverse transcription Sybr green Tnf α

Corresponding Organization : University of Cologne

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Variable analysis

independent variables

Method of RNA extraction (RNeasy Mini Kit or Tri Reagent)

dependent variables

Expression levels of genes (iNOS, Arg I, TNF-α, IL-10, TGF-β, MMP-2, MMP-9, Collagen-1, Collagen-3, IL-6, Fizz1/RELM α, Ym-1, SPHK1, LIGHT, MertK)

control variables

Endogenous control (18S RNA) for normalization of gene expression
Thermal cycling conditions (2 min 50°C, 2 min 95°C, 40 cycles of 1 s 95°C and 30 s 60°C, and 4°C hold)

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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