Minikit rneasy columns

Total RNA was isolated from purified B cells using MiniKit RNeasy columns (QIAGEN) with a DNase-I treatment step. One microgram of DNA-free RNA was reverse transcribed to cDNA using Omniscript RT (QIAGEN). TaqMan gene expression assays containing FAM dye-labeled TaqMan MGB probe was used for mouse S1pr1 (Mm02619656_s1), RARα (Mm01296312_m1), RARβ (Mm01319677_m1), and RARγ (Mm00441091_m1) in multiplex with primer-limited assays for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) endogenous control containing VIC/MGB probes. Real-time quantification was performed using TaqMan gene expression master mix on a Bio-Rad CFX96 optical reaction module on a C1000 thermal cycler. Data were analyzed using CFX Manager Software (Bio-Rad).

Total RNA was isolated by using MiniKit RNeasy columns (QIAGEN) with a DNase-I treatment step. One microgram of DNA-free RNA was reverse transcribed to cDNA using Omniscript RT (QIAGEN, Hilden, Germany) following manufacturer’s instructions. TaqMan gene expression assays containing FAM dye-labelled TaqMan MGB probe were used for human HNF4a (Hs00230853_m1), SERPINA (Hs00165475_m1), Toll-like receptor (TLR) 2 (Hs01872448_s1), TLR4 (Hs00152939_m1), TLR5 (Hs01920773_s1), TLR7 (Hs01933259_s1) and TLR9 (Hs00370913_s1) in multiplex with VIC dye-labelled TaqMan probe for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as endogenous control. Real-time quantification (40 cycles) was performed using TaqMan Gene Expression Master Mix or Power SYBR Green PCR Master Mix (ThermoFisher, Waltham, MA, United States) on a Bio-Rad CFX96 optical reaction module on a C1000 thermal cycler following manufacturer’s instructions. Data were analysed using CFX Manager 3.1 Software (Bio-Rad, Hercules, CA, USA).