THE JOURNAL OF EXPERIMENTAL BIOLOGY 1266 growth performance (El-Sayed and Kawanna, 2008;Yan and Wang, 2010; Chen et al., 2011): (1) initial mass (g), wet mass of fish at the beginning of culture; (2) final mass (g), wet mass of fish at the final of culture; (3) daily mass gain (DMG), calculated as (final mass-initial mass)/culture days; (4) coefficient of variation (CV) for final mass, calculated as mean standard deviation of the final mass/mean final mass; (5) feed conversion ratio, calculated as feed consumption/mass gain; and (6) feed intake, calculated as 100×feed consumption/[days×(final mass+initial mass)/2)].
Quantitative PCRTotal RNAs were extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA), and miRNAs were extracted using the miRNeasy kit (Qiagen, Gaithersburg, MD, USA). Real-time PCR for IGF-1 was performed using SYBR Green PCR mixture (Takara Bio, Otsu, Shiga, Japan) in a MyiQ5 Real-time PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). Gene expression was normalized relative to the housekeeping gene (β-actin). The primer sequences are as follows: IGF-1, 5′-GTCTGTGGAGAGCGAGGCTTT-3′ (forward), 5′-CACGTGAC -CGCCTTGCA-3′ (reverse); β-actin, 5′-GTCCCTGTACGC-CTCTGGTCG-3′ (forward), 5′-GCCGGACTCATCGTACTCCTG-3′ (reverse). The relative amount of miRNA was detected using stemloop PCR, and U6 RNA was detected as the internal control. Relative gene or miRNA expression was determined using the comparative C t method, which is also referred to the 2 -ΔΔCt method (Chen et al., 2005;Schmittgen and Livak, 2008).
Silencing of miR-206 in vivo using the antagomir methodThe antagomirs used in the study are single-stranded RNAs, which consist of 21-23 nucleotides modified as follows: antagomir-133a, CsAsGCUGGUUGAAGGGGACCsAs As As-Chol-3′; antagomir-206, CsCsACACACACUUCCUUACAUUs CsC sAs-Chol-3′; and mismatch antagomir, CsAsCGGUUCCAGGCACUGUsG sUs AsChol-3′. All nucleotides are 2′-OMe-modified; the 's' represents a phosphorothioate linkage; 'Chol' represents cholesterol linked through a hydroxyprolinol linkage. They were deprotected, desalted and purified by high-performance liquid chromatography. Antagomir was dissolved in phosphate-buffered saline before injection. Tilapia weighing approximately 1g received a 0.02ml tail-vein injection of saline or antagomir at a dose of 60mgkg -1 body mass twice a week. Tissues were harvested, snap-frozen and stored at -80°C.Cell culture and 3′-UTR luciferase reporter assay HEK 293T cells were obtained from the American Type Culture Collection (Rockville, MD, USA). They were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum, 100Uml -1 penicillin, 100μgml -1 streptomycin and 250ngml -1 amphotericin B, and maintained at 37°C in a humidified 5% CO 2 incubator.To generate the 3′ UTR luciferase reporter construct, the full length of the 3′ UTR from IGF-1 was cloned into the downstream of the firefly luciferase gene in pGL3-control vector (Promega, Madison, WI, USA). The QuikChange site-directed mutagenesis kit (Stratagene, La Jolla...