Circadian Rhythm Analysis of Transgenic Zebrafish

Adult homozygous Tg(aanat2:EGFP-ΔCLK) fish were raised in a temperature-controlled recirculation water system under 12-hr:12-hr LD cycles, and transferred to DD at the end of the light period prior to sampling. Pineal glands were sampled at 4-hr intervals throughout two daily cycles under DD at 12 time points corresponding to circadian time (CT) 14, 18, 22, 2, 6, 10, 14b, 18b, 22b, 2b, 6b and 10b (Fig 4A), as previously described [42 (link)]. A pool of 16 pineal glands was collected at each time point. In addition, two control pools of 14 pineal glands were collected from Tg(aanat2:EGFP) fish at time points corresponding to CT2 and CT14b. Fish were anesthetized in 1.5 mM Tricane (Sigma-Aldrich) and decapitated. Fluorescent pineal glands were selectively removed under a dissecting microscope (Olympus SZX12) equipped with filters for excitation (460–490 nm) and emission (510–550 nm) of EGFP. Total RNA for mRNA analysis was isolated using RNeasy Lipid Tissue Mini Kit (Qiagen).