Agilent bioanalyzer rna total eukaryote 2100 nano

Changes in relative gene expression were analysed by RT-PCR. EA.hy926 cells were cultured in 6-well plates (Corning TM, Corning, NY) (cell density of 6 x 10⁵ cells/mL) with FAs for 48 h followed by incubation with TNF-α (1 ng/mL) for 6 h. Taqman Gene Expression Primers (ThermoFisher Scientific, Waltham, MA, USA) were used to determine the expression of NFκB1 (Hs00765730_m1), NFκBIA (Hs00355671_g1), IκBKB (Hs00233287_m1), PTGS2 (Hs00153133_m1), and IL-6 (HS00985639_m1). Total RNA was extracted from the cells using the ReliaPrep RNA cell Miniprep System (Promega, Southampton, UK). RNA quantity and quality were analysed by NanoDrop. Analysis of RNA using an Agilent Bioanalyzer (RNA Total Eukaryote 2100 Nano) was performed to determine RNA integrity through RIN scores. cDNA was synthesised from total RNA using GoScript Reverse Transcriptase (Promega). Housekeeping reference genes were determined using a geNorm Kit (Primerdesign, Camberley, UK). Quantification of relative gene expression was analysed using YWHAZ, (Hs01122445_g1), CYC1 (Hs00357717_m1), and RPL13A (Hs04194366_g1) as housekeeping genes.

Changes in the relative gene expression were analysed by RT-PCR. The EA.hy926 cells were cultured in 6-well plates (Corning, Corning, NY, USA) (cell density of 6 × 10⁵ cells/mL) with FAs for 48 h and then without FAs for 6 h. Taqman Gene Expression Primers (Thermo Fisher Scientific, Waltham, MA, USA) were used to determine the expression of NFκB1 (Hs00765730_m1), NFκBIA (Hs00355671_g1), IκBKB (Hs00233287_m1), PPARα (Hs00947536_m1), PTGS2 (Hs00153133_m1) and IL-6 (HS00985639_m1). Total RNA was extracted from the cells using the ReliaPrep RNA cell Miniprep System (Promega, Southampton, UK). RNA quantity and quality were analysed by NanoDrop. RNA integrity was assessed as RIN score using an Agilent Bioanalyzer (RNA Total Eukaryote 2100 Nano). cDNA was synthesised from total RNA using GoScript Reverse Transcriptase (Promega, Southampton, UK). Housekeeping reference genes were determined using a geNorm Kit (Primerdesign, Camberley, UK): YWHAZ (Hs01122445_g1) and RPL13A (Hs04194366_g1) were used as housekeeping genes to determine quantitative gene expression.