Inflammatory cytokines and receptors pathway

Total RNA was isolated from cell lines using GenElute Mammalian Total RNA Purification Miniprep Kit (Sigma-Aldrich, Inc,), according to manufacturer’s instructions. Pure RNA was quantified at NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA).
Complementary DNA (cDNA) synthesis was performed starting from 500 ng of total RNA, using PrimeScript RT Master Mix (Takara Bio, Inc., Kusatsu, Japan). The reaction was incubated at 37°C for 15 min and inactivated by heating at 70°C for 10 s. cDNA was amplified by real-time quantitative polymerase chain reaction (PCR) using the ViiA™ 7 System (Applied Biosystems, Foster City, CA, USA).
All PCR reactions were performed in a 20 µL volume. Each reaction contained 10 µL of 2× qPCRBIO SYGreen Mix Lo-ROX (PCR Biosystems, Ltd, London, UK), 400 nM concentration of each primer, and cDNA.
Custom primers belonging to the “Inflammatory Cytokines and Receptors” pathway were purchased from Sigma-Aldrich, Inc. All experiments were performed including non-template controls to exclude reagents contamination. PCR was performed including two analytical replicates.
The amplification profile was initiated by 10 min incubation at 95°C, followed by two-step amplification of 15 s at 95°C and 60 s at 60°C for 40 cycles. As final step, a melt curve dissociation analysis, was performed.

Total RNA was isolated from cell lines using GenElute mammalian total RNA purification Miniprep Kit (Sigma-Aldrich) according to the manufacturer’s instructions. Pure RNA was quantified at NanoDrop 2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA).
Complementary DNA (cDNA) synthesis was performed starting from 500 ng of total RNA, using PrimeScript RT Master Mix (Takara Bio Inc., Kusatsu, Japan). The reaction was incubated at 37°C for 15 min and inactivated by heating at 70°C for 10 s. cDNA was amplified by real-time quantitative polymerase chain reaction (PCR) using the ViiA™ 7 System (Applied Biosystems, Foster City, CA, USA).
All PCR reactions were performed in a 20-µL volume. Each reaction contained 10 µL of 2× qPCRBIO SYGreen Mix Lo-ROX (PCR Biosystems, London, UK), 400 nM concentration of each primer, and cDNA.
Custom primers belonging to the “Inflammatory Cytokines and Receptors” pathway were purchased from Sigma-Aldrich. All experiments were performed including non-template controls to exclude reagents contamination. PCR was performed including two analytical replicates.
The amplification profile was initiated by 10 min incubation at 95°C, followed by two-step amplification of 15 s at 95°C and 60 s at 60°C for 40 cycles. As a final step, a melt-curve dissociation analysis was performed.