Figure 1
High content image analysis reveals the impact of miR-124 on primary motor neuron morphology. (a) Values for nine individual miRNAs, all transfected at 0.5 ng/µl, to mouse primary motor neurons. miRNA expression levels displayed on the Y-axis as 40 minus qPCR cycle threshold (40-Ct), on a Log2 scale. All miRNAs were significantly overexpressed. (b) A diagram describing the method: Spinal motor neurons were isolated from E13.5 mouse embryos and seeded on a 384 multiwell plate. Culture was transfected with different miRNA mimics using Bravo automated liquid handling robot. 72 hrs later, cells were fixed, stained with anti Tuj1 antibody and DAPI. Two fluorescent micrographs were captured per well (ImageXpress Micro and MetaXpress2 software, Molecular Devices). (c) Cell numbers (Cell), neurite outgrowth per cell (outgrowth) and number of branches per cell (branches), were quantified with serial doses of the stress-inducing agent, Sodium Arsenite (15, 30 and 60 µM, for 60 minutes). See Methods and Sup. Figure 2. (d) None of the nine miRNAs tested influenced cell numbers. miR-124 was the only miRNA to reduce mean axonal outgrowth per cell and mean number of branches per cell. 500 Tuj1+ neurons quantified per field, 2 fields/well and 6 wells per treatment in five independent experimental repeats. Data collected from >30,000 Tuj1+ neurons per treatment. Averages \(\pm \) SEM, Student’s t-test. *P-value < 0.05.
