Viia7 real time pcr thermocycler

Total RNA was obtained and isolated using RNeasy Mini Kit (Qiagen, Venlo, Netherlands). 0.5 μg of this RNA were reversely transcribed using Moloney Murine Leukemia Virus reverse transcriptase, oligo (dT) and random decamers (Ambion, Carlsbad, CA). Two microliters of the reaction were used as a template for the qPCR amplification reaction (LightCycler 480SYBER Green I Master Mix, Roche) in a ViiA 7 Real-Time PCR thermocycler (Applied Biosystems). Quantitative data was normalized to β-actin expression. All primers used are available in Table S2.

T_eff frozen in Qiazol were defrosted and RNA was isolated and purified via chloroform extraction using the miRNeasy Micro Kit, following the manufacturer’s protocol for isolation of total RNA from small samples [Qiagen]. RNA concentration was quantified using a Nanodrop spectrophotometer [Thermofisher Scientific]; 100 ng of total RNA per sample was reverse-transcribed using the RevertAid First Strand cDNA Synthesis Kit and random hexamer primers according to the manufacturer’s protocol. Quantitative polymerase chain reaction [qPCR] was performed using a ViiA 7 real-time PCR thermocycler [Applied Biosystems] in a final volume of 10 µl, using the 2 x Maxima probe/ROX qPCR Master Mix [Thermofisher Scientific] and the cDNA equivalent of 5 ng total RNA per sample. Detection of transcripts for pro-inflammatory cytokines was carried out using FAM-labelled 20 x TaqMan probe assays [Thermofisher Scientific] for IFNG [assay ID: HS00989291_m1] and TNF [assay ID: HS00174128_m1] and relative expression of each gene was calculated using the comparative cycle threshold [Ct] method [2^-ΔCt] using GAPDH [assay ID: HS99999905_m1] as the endogenous reference control. Reactions were run in duplicates and the average Ct value for each reaction was used for analysis.