RNA sequencing and RT-qPCR for differentially expressed genes in otoferlin depleted larvae. (A) Black bar graphs represent relative mRNA expression changes of the upregulated and downregulated genes derived from transcriptomic analysis (no. of biological reps. =4, each replicate consists of 15 pooled embryos). Grey bars represent qRT-PCR data of mRNA expression changes of genes in larval otoferlin mutant zebrafish at 96 hpf (no. of biological reps. = 4, each replicate consists of 30 pooled embryos). Each gene is normalized with respect to the corresponding controls. qRT-PCR, ΔΔCt values were calculated by comparing ΔCt values (normalized to beta-actin) to the mean ΔCt for each gene. Data were analyzed by Wilcoxon’s test with standard 5% significance level. Abbreviations otof a: Otoferlin a, otof b: Otoferlin b, rtn4rl2a: Reticulon 4 Receptor like 2a, rtn4rl2b: Reticulon 4 Receptor like 2b, s100s: S100 Calcium Binding Protein S, pvalb9: Parvalbumin 9. (B) Rescue of repressed transcripts in otoferlin depleted larvae. Bar graphs represent relative mRNA expression changes of transcripts in otoferlin depleted larvae at 96 hpf (denoted otoAB KD, black bars) and rescue of the transcripts (denoted (m) otof, grey bars) in morphants coinjected with full length otoferlin rescue construct. Each gene is normalized with respect to the corresponding controls (white bars). ΔΔCt values were calculated by comparing ΔCt values (normalized to beta-actin) to the mean ΔCt for each gene. Data were analyzed by t-test. For pval9b and rtn4rl2b groups both otoferlin depleted and rescue samples showed statistically significant changes in expression compared to controls (p-value < 0.05). For rtn4rl2a significant repression in expression for the KD were found (p-value < 0.05), and there was restoration of repressed transcripts in the rescue larvae. (C) Expression of s100s, pval9b, rtn4rl2a, rtn4rl2b at 24, 48 and 72 hpf in control and otoferlin depleted zebrafish larvae (n = 4 p < 0.05). For (A–C), error bars are s.e.m.
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