Quantifying Neuronal miRNA Expression

Dissociated hippocampal and cortical neurons from embryonic E18 rat embryos were prepared as described (Fivaz and Meyer, 2005 (link); Kaech and Banker, 2006 (link)). 500,000 neurons were seeded per well in a poly-L-lysine-coated 12-well cell culture plate and transduced with lentiviruses to overexpress gfp, miR-196a, miR-27b, or miR-324 3 h after plating or electroporated with miRIDIAN hairpin inhibitors against miR-27b-5p or miR-324-5p or a negative control inhibitor. Neurons were cultured in Neurobasal media with B27 supplement. Total RNA was extracted from DIV8-14 neurons using Sepasol RNA I Super G according to the manufacturer's instructions (Nacalai Tesque) and 500 μl of Sepasol was added per well. Five nanograms of total RNA was reverse transcribed using the Taqman miRNA RT kit and RT primers specific for Y1 scRNA (control), miR-196a-5p, miR-27b-5p, miR-324-5p from the Taqman miRNA assay (Applied Biosystems). miRNA-specific qPCR probes and Taqman 2× PCR master mix (Applied Biosystems) were used to detect levels of mature miRNAs. qPCR reactions were set up in triplicates according to the manufacturer's protocol and reactions were carried out on the Bio-Rad CFX-96 machine (Bio-Rad). miRNA levels were normalized to the Y1 scRNA.