Fig. 4. Qualitative and quantitative analysis of miR-375-3p expression level in the cervical spinal cord of the wobbler (WR) in comparison to wild type (WT) mouse. (A) Expression levels of miR-375-3p from presymptomatic to stationary phase of wild type (WT) and wobbler (WR) spinal cord were investigated by qPCR. Significant deregulation of miR-375-3p was detected at all developmental stages. For relative quantification of miR-375-3p expression, the 2^-ΔΔCt method was conducted using the housekeeping gene U6 for normalization. Data are provided as means ± SEM. Data were tested for significance using Student's t-test. Significant differences are indicated by **p<0.01, ***p<0.001. n = 6-10. (B) Exemplary overview of in situ hybridization with miR-375-3p-probe in a cross section of the cervical spinal cord of wild type (WT) and wobbler (WR) mice at the developmental stage p20 and p40. There are no visible staining pattern differences between WT and WR. Scale bar = 1mm. (C) Cross section of the cervical spinal cord of a wild type mouse at p40. Exemplary large-scale picture of the anterior horn after in situ hybridization with miR-375-3p-probe. Arrows indicate motor neurons with a clearly visible prominent staining for miR-375-3p. Scale bar = 500 µm. All pictures were taken with a light microscope (Olympus microscope BX61VS, Japan) and a 20x objective (UPlanSApo 20x/0.4, Olympus, Japan). For counterstaining Nuclear Fast Red was used.