Fig. 6.
Determining Caprin-1 mRNA expression in hair cells following neomycin treatment. (A) Mixed-cell samples of outer hair cells and Deiters' cells were collected from cochlear explant cultures treated with 1 mM neomycin for 0, 3 or 12 hours, in three separate experiments. Caprin-1 and Pou4f3 mRNA expression levels were calculated relative to the expression at 0 hours and normalised to expression of the cell markers p27kip1 and Myo7a, respectively. This adjustment is made to normalise for differences in the supporting cell:hair cell ratio in these samples. Caprin-1 is expressed in supporting cells and hair cells, whereas Pou4f3 is only in the hair cells. (B) Individual hair cells were collected from five different experiments. After determining that samples were hair-cell-specific (using Myo7a expression and p27kip1 exclusion), Caprin-1 and Pou4f3 levels were measured by qPCR. Expression levels were calculated relative to expression of an individual cell at 0 hours to determine the relative quantification (RQ) value. Individual hair cell results are plotted (each symbol represents a different experiment) as well as the mean value for each time point (grey bar). The s.e.m. for each experiment was as follows: Caprin-1, 0 hours ±0.98, 3 hours ±1.76, 12 hours ±5.80; Pou4f3 0 hours ±0.42, 3 hours ±0.31, 12 hours ±0.90. (C) Individual hair cells collected from the culture in Experiment 1 (see A and dark blue triangles in B). Expression levels are calculated relative to the expression at 0 hours and are plotted on a logarithmic scale. All error bars represent the 95% confidence interval.