Glomax r96 microplate luminometer

Strains were pre-lysed in a lysis buffer composed of 2 mg lysozyme (Sigma-Aldrich, Dorset, UK), 120 U mutanolysin (Sigma-Aldrich), 400 μg RNase A (Qiagen, Manchester, UK), 20 μL proteinase K (>600 mAU/mL) and 15 μL of overnight culture. Cell suspensions were incubated for 1 h at 37 °C, 2 h at 56 °C followed by 1 h at 80 °C. DNA was extracted using the QIAsymphony SP system and the QIAsymphony DSP mini kit (Qiagen) according to the manufacturers’ recommendations. DNA concentrations were measured using the Quant-iT dsDNA Broad-Range Assay Kit (Life Technologies, Paisley, UK) and GloMaxR 96 Microplate Luminometer (Promega, Southampton, UK). For sequencing preparation, a Nextera XT DNA Library Preparation Kit (Illumina, San Diego, CA, USA) was used followed by sequencing using a HiSeq 2500 System (Illumina) and the 2 × 100-bp paired-end mode.

Rat hippocampal (DIV5 to DIV11) and cortical neurons (DIV4 to DIV6) from three independent biological replicates were transfected respectively in technical triplicates with 200 ng or duplicates with 100 ng of Firefly and Renilla luciferase constructs. At the indicated DIV after transfection, cells were pre-incubated in APV as described before, further stimulated with BDNF for 6 h and lysed in passive lysis buffer (Promega). For pmirGLO reporter transfections, DIV5 rat cortical cells from four independent biological replicates were transfected with 500 ng pmirGLO constructs and with 500 ng pri-miR constructs in technical duplicates and 48 h later cells were lysed.
The activity of Firefly and Renilla luciferases was measured with a GloMax R96 Microplate Luminometer (Promega). Averages of Firefly/Renilla activity ratios from miR-134 reporters and mutants were further normalized with average ratios obtained from the activity of empty luciferase vectors, subjected to the same drug treatment/transfection conditions.

Primary rat cortical neurons were transfected in triplicates with 100 ng pmiRGlo luciferase reporters alone or with 10 nM miRNA mimics or LNA miRNA inhibitors. Neurons were lysed in 5× Passive Lysis buffer (Promega) 2 days after transfection, and dual‐luciferase assay was performed using homemade reagents as previously described 75 on the GloMax R96 Microplate Luminometer (E1941, Promega).