Mircury lna inhibitor control

Dulbecco’s modified Eagle’s medium (DMEM), ISOGEN II and human recombinant IL-1β were purchased from Wako (Tokyo, Japan). Human recombinant TNF-α was purchased from R&D Systems (Minneapolis, MN, USA). Fetal calf serum (FCS), Lipofectamine 2000, penicillin and streptomycin, and TrypLE™ Express were purchased from Invitrogen (Carlsbad, CA, USA). The PrimeScript RT reagent kit and SYBR Premix Ex Taq™ II were obtained from Takara (Tokyo, Japan). The miRNeasy Mini Kit was purchased from Qiagen (Valencia, CA, USA). The Mir-X miRNA First-Strand Synthesis Kit and SYBR Advantage qPCR Premix were purchased from Clontech (Mountain View, CA, USA). Expression plasmid for miRNA (pEZX-MR04) was obtained from GeneCopoeia (Rockville, MD, USA). miRCURY LNA inhibitor and miRCURY LNA inhibitor Control were purchased from Exiqon (Woburn, MA, USA). Anti-rabbit IgG (whole molecule)-peroxidase antibody produced in goat was from Sigma-Aldrich Japan (Tokyo, Japan). ELC plus Western Blotting Detection Reagents were purchased from GE Healthcare UK Ltd. (Buckinghamshire, UK). All chemicals used were of analytical grade.

At DIV8, neurons were transfected with either complementary sequence based locked nucleic acid inhibitors of miR-19a or miR-539 (miR-19a, #410118-00, Exiqon; miR-539, #MC11336, Exiqon), or a scrambled control (miRCURY LNA Inhibitor Control, #199004-00, Exiqon) using Lipofectamine 2000. The following day, protein lysates were collected.

Antisense oligonucleotides form duplexes and sequester the respective miRNA, leading to inhibition of the target miRNAs [53 (link),89 (link),90 (link)], hence, the name “miRNA inhibitors”. The cellular uptake of miRNA inhibitors is an active process that does not require carriers or transfection reagents. Modifications of the nucleic acids as 2’-O-methyl, 2’-O-methoxyethyl and LNAs increase cellular stability. ALI cultures of F508del HBE cells were treated with 50 nM LNA-antisense probe (miRCURY LNA™ Power Inhibitor hsa-miR-145-5p; Exiqon) or control (miRCURY LNA™ Inhibitor Control), added to the basolateral medium every other day for 8 days. CFTR correctors were added daily for 48 h, and TGF-β1 or vehicle control was added for 24 h before experiments.
ALI cultures of HBE cells were treated with 5 nM miRCURY LNA™ Power Mimic (has-miR-145-5p; Exiqon), referred to as miR-145 analog or analog control (miRCURY LNA™ Power Negative control), and added to the basolateral medium with HiPerFect Transfection reagent (Qiagen) for 48–72 h. Following the above treatments with either miRNA inhibitors or analog, cells were lysed and mRNA and miRNA levels were quantified by qRT-PCR.