Nine fish from each group were randomly selected to detect the miR-430 expression after microinjection. The total RNAs were extracted using TRIzol regent (Takara, Tokyo, Japan) according to the manufacturer’s instructions. mRNA reverse transcriptions were performed with equal amounts of total RNA (1 µg) from each sample as a template with Quantitect Reverse Transcription Kit (Takara) following the manufacturer’s protocol. The stem-loop qRT-PCR of microRNAs was performed. Oligo dT/Random primers were replaced with miR-430s stem-loop RT primers in a TAKARA Reverse Transcription Kit (Table S5 ). The qRT-PCR was performed with the SYBR Premix Ex TaqTM II kit (Takara) on a Chromo 4 Real-Time Detection System (MJ Research, Hercules, CA, USA) following the manufacturer’s protocols in the previously reported method [10 (link)]. The gene-specific primers for each gene are listed in Table S5 . The relative expression levels of miR-430s were analyzed using the comparative ΔΔCt method, and they were normalized with U6 (Figure 5 D). All experiments were performed in triplicate.
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