Myogenic (Pax3, MyoD1, Myf5, Myf6, Desmin, and MYH2) and cytokine (IL6, TNF, IL12A, IL1B, IFNG, IL10, IL4, TGFB1 and TGFB2) (all from Bio-Rad, Foster City, CA, USA; Supplementary Table S2 ) gene expression was investigated by Reverse Transcription quantitative polymerase chain reaction (RT-qPCR) (Supplementary Material ). Experiments were run in duplicate. Data were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression (reference gene) applying the geNorm method [56 (link)] and using CFX Manager software (M < 0.5). Fold changes were determined by 2−ΔΔCt method and presented as relative levels versus untreated cells.
Tumor necrosis factor (tnf)
Manufactured by Bio-Rad
Sourced in United States
TNF is a lab equipment product manufactured by Bio-Rad. It is a device used for the detection and quantification of tumor necrosis factor (TNF) in samples.
Automatically generated - may contain errors
2 protocols using tumor necrosis factor (tnf)
1
Myogenic and Cytokine Gene Expression
2
Quantitative PCR analysis of key genes
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cDNA was synthesized from 1 μg total RNA using the iScript™ cDNA Synthesis kit (Bio-Rad; Cat. No. 1708891) in a 20 μL reaction volume, according to the manufacturer’s instructions. Gene expression findings of key genes identified by microarray and RT2 PCR arrays were validated by qPCR using a StepOnePlus™ real-time PCR system (Thermo Fisher Scientific), wet-lab validated PrimePCR™ Gene Expression Probe assays (Bio-Rad) and were conducted following MIQE guidelines [25 (link)]. Genes assayed by qPCR were TNF (unique assay ID dHsaCPE5190842; Bio-Rad), TLR4 (dHsaCPE5030581), CCL5 (dHsaCPE5050154), ZFP36 (dHsaCPE5191899), EDN1 (dHsaCPE5053386) and ITLN1 (dHsaCPE5041777). The reference gene was RPLP0 (dHsaCPE5031575). Samples were assayed in a minimum of biological triplicates, assayed in technical triplicate, and data were analysed using the comparative Ct method (ΔΔCt), with results reported as the fold-change in gene expression (2-ΔΔCt) relative to the DMEM/10% LB control.
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