Quantitative PCR

Total RNA was isolated from ventral bone and cartilage of juvenile golgb1^Q2948X and golgb1^X3078 genotyped fish (60 and 63 dpf, respectively, n=3 per genotype) using RNeasy mini kit (74104, Qiagen). Subsequently, a reverse transcriptase reaction was performed by using Superscript IV (18091050, Thermo Fisher). Zebrafish galnt3 ({"type":"entrez-nucleotide","attrs":{"text":"XM_009300463.2","term_id":"1040699384","term_text":"XM_009300463.2"}}XM_009300463.2) coding sequence was confirmed by multi-species nucleotide BLAST (NCBI) leading to galnt3 forward, 5′-TCCTTCAGAGTGTGGCAGTG and reverse, 5′-TGATGGTGTTGTGGCCTTTA primers. gapdh was used as a reference gene (forward, 5′-TGTTCCAGTACGACTCCACC and reverse, 3′-GCCATACCAGTAAGCTTGCC). Quantitative real-time PCR (qPCR) reactions (quadruplicates per individual) using DyNAmo HS SYBR green (F410L, Thermo Fisher) with PCR cycles (40 times) of 95°C for 25 s, 57.5°C for 30 s and 70°C for 45 s, followed by a standard melt curve were applied (QuantStudio3, Applied Biosystems).