Mir nc oligonucleotides

IEC18 and Caco-2 cells were used at 60–80% confluency. Cells were cultured in 24-well plates with DMEM supplemented medium with heat-inactivated FBS (10% v/v), 100 IU/ml penicillin, 0.1 mg/ml streptomycin, 2.5 µg/ml amphotericin and 2 mM L-glutamine, in a humidified 5% CO₂ atmosphere at 37 °C. Lipofectamine RNAiMAX (ThermoFisher, Waltham, MA, USA) was used to transfect cells lines IEC18 and Caco-2 with hsa-miR-146a-5p mirVana mimic or nontargeting negative control (miR-NC) oligonucleotides, at 30 and 60 nM respectively (Life Technologies, Carlsbad, CA) according to the manufacturer’s instructions. The mirVana mimics (Ambion/ThermoFisher) are double-stranded oligonucleotides mimicking mature microRNA.

To study the effect of miR-144-3p on Huh7 and 1.1B4 cell viability and apoptosis, cells were transfected using lipofectamine for 24 h and 48 h with the specific miR-144-3p mimic mirVana™ or scramble miR (miR-NC) as a control, and subsequently studied using the XTT cell proliferation and TUNEL assays. Cell viability was studied after 48 and 24 h of transfection with miR-144-3p mimic or miR-NC.
Cells were seeded in a 48- or 98-well tissue-culture plate and cultured for 1–2 days. Transfection was performed when the cells were 60–80% confluent. Lipofectamine RNAiMAX (Invitrogen) was used to transfect cells with a final concentration of 30 and 60 nM miR-144-3p mimic (mirVana™) or non-targeting negative control (miR-NC) oligonucleotides (Life Technologies) according to the manufacturer’s instructions. Serum-free medium Opti-MEM^® was used for transfection.
Transfection efficiency of miR-144-3p (after 48 h of transfection) was assessed by RT-qPCR normalized to U6 small RNA and to cells treated, or not, with miR-NC.