Mircury lna microrna inhibitor control

LCLs 3A-REV were electroporated with 50 nM of LNA anti-miR-221 oligonucleotide (hsa-miR-221 miRCURY LNA, Exiqon), LNA anti-miR-222 oligonucleotide (hsa-miR-222 miRCURY LNA, Exiqon) or scrambled oligonucleotide (miRCURY LNA microRNA inhibitor control, Exiqon) using a Bio-Rad Gene Pulser I (270V, 960μF). After 48 h, dead cells and debris were removed by layering the cells over 3ml Ficoll-plaque (GE Healthcare). Live cells were then collected washed in PBS and extraction was performed as previously described for Western blotting.

For mature miR-195-5p or miR-107 expression, we used Pre-miRNA Precursor-Negative Control (Ambion) and Pre-miRNA195-5p (Ambion) or Pre-miRNA107 at final concentration of 5nM. For miR-195-5p and miR-107 depletion we used miRCURY LNA microRNA inhibitor control (Exiqon) and hsa-miR-195-5p miRCURY LNA (Exiqon) or hsa-miR-107 miRCURY LNA (Exiqon) at final concentration of 10nM. U87MG cells were transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturer’s instructions. For miRNAs depletion experiments, after 48 h of transfection cells were treated with IC20 BRV or IC20 LCM for 48 h.

Human LHCN cells were transfected with different miRNA inhibitors (miRCURY LNA microRNA Inhibitors) or a scrambled control (miRCURY LNA microRNA Inhibitor Control) from Exiqon (Vedbaek, Denmark) or/and pre-miRNA precursors and precursor scrambled control (mimics) from Ambion (Life Technologies) as follows: The transfection mix for a 96-well plate format contained 10 μl Opti-MEM I Reduced Serum Medium, GlutaMAX Supplement (Life Technologies) per well with final miRNA inhibitor concentrations of 25 nM, 50 nM, or 100 nM, or final miRNA mimic concentrations of 5 nM, 25 nM, or 50 nM as well as Lipofectamine RNAiMAX transfection reagent (Life Technologies). The transfection mix was incubated for 20 minutes at room temperature and added to the well before plating 90 μl of cell suspension. 24 hours after transfection, the growth medium was replenished or the differentiation medium with TNF-α or IGF1 was added, respectively.