One-cell stage embryos were injected with 2.3 nL of 250-500 μM duox MO1 (5'-AAGCGTCACTTACTATAATGTTGGA-3', Gene Tools) or MP (5'-TCCCTTTTAGAATTTACCTTGCCGA-3', Gene Tools)2 (link) together with 200 μM p53 morpholino (5'-ATGCTCAACTATAATGTTGGACATT-3', Gene Tools)38 (link) diluted in water. P53 morpholino co-injection with duox MO1/MP served to suppress potential, pleiotropic morpholino-associated toxicity as previously described 2 (link),38 (link).
To assess the efficacy of the injected morpholino, ~100 larvae (2 dpf) were dissociated in 500 μL of TRIzol reagent (Invitrogen, 15596026) using RNase-free disposable pellet pestles (Fisherbrand, 12-141-364). Total RNA was extracted as per manufacturer’s instructions. Contaminating DNA was removed using the TURBO DNA-free Kit (Invitrogen, AM1907). Knockdown was confirmed by using the OneStep Ahead RT-PCR Kit (Qiagen, 220211) with the following primers: duox forward 5’-ACACATGTGACTTCATATCCAG-3’ and duox reverse 5’-ATTATTAACTCATCCACATCCAG-3’.
To assess the efficacy of the injected morpholino, ~100 larvae (2 dpf) were dissociated in 500 μL of TRIzol reagent (Invitrogen, 15596026) using RNase-free disposable pellet pestles (Fisherbrand, 12-141-364). Total RNA was extracted as per manufacturer’s instructions. Contaminating DNA was removed using the TURBO DNA-free Kit (Invitrogen, AM1907). Knockdown was confirmed by using the OneStep Ahead RT-PCR Kit (Qiagen, 220211) with the following primers: duox forward 5’-ACACATGTGACTTCATATCCAG-3’ and duox reverse 5’-ATTATTAACTCATCCACATCCAG-3’.