Computational protocol: Antioxidant and Anti-Inflammatory Effects in RAW264.7 Macrophages of Malvidin, a Major Red Wine Polyphenol

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Protocol publication

[…] Total RNA was extracted from RAW 264.7 cells using TRIZol reagent (Sigma-Aldrich), according to the manufacturer’s protocol. RNA (1 µg) was reverse-transcribed with MMLV RT (RevertAid™ first-strand cDNA synthesis kit, Fermentas, Burlington, Ontario, Canada) for 1h at 42°C; final volume was 20 µl. cDNA (1 µl) was used for real-time PCR amplification on a Bio-Rad Mini Opticon (MJ Mini) machine. PCR was conducted over 45 cycles of 95°C for 15 s, 55°C for 30 s, and 72°C for 45 s; three-step thermal cycling preceded by an initial 95°C for 7 s using the iQ SYBR Green Supermix kit (Bio-Rad, Hercules, CA, USA). PCR was performed using the following primers:MKP-1 forward, 5′-GCATCCCTGTGGAGGACAACC-3′;MKP-1 reverse, 5′-TCCAGCATCCTTGATGGAGTCTATG-3′;β-Actin forward, 5′-GCCACCAGTTCGCCATGGAT-3′;β-Actin reverse, 5′-GCTTTGCACATGCCGGAGC-3′.Statistical analysis of relative expression of the target gene based on comparative threshold values with efficiency correction was made with the Relative Expression Software tool (Bio-Rad CFX Manager Software), and was normalized to the housekeeping gene β-Actin. All experiments were repeated three times. […]

Pipeline specifications

Software tools REST, CFX Manager
Application qPCR
Diseases Cardiovascular Diseases, Diabetes Mellitus, Hypertension
Chemicals Adenosine Diphosphate, Oxygen, Poly Adenosine Diphosphate Ribose, Ribose, Polyphenols