Figure 1.

mper2 mRNA is degraded in a phase dependent manner. (A) Oscillation of endogenous mper2 mRNA was measured by quantitative real-time PCR in NIH 3T3 cells. NIH 3T3 cells were treated with 100 nM dexamethasone twice for 2 h at −24 and 0 h to synchronize the circadian oscillation. Total RNA (1 μg) from each time point was subjected to quantitative real time RT–PCR with specific oligonucleotides for mper2 and mtbp (TATA box binding protein) as a normalizing control. All results are expressed as the mean ± SEM of three independent experiments. (B) mRNA degradation kinetics were measured during both declining (closed triangles) and rising (closed squares) phases in the 2nd day after dexamethasone treatment (shaded box in Figure 1A). Cells were treated with 5 μM actinomycin D at 32 h, during the rising phase, and at 40 h, during the declining phase, and then harvested at 2 h intervals. Total RNA (1 μg) was subjected to quantitative real time RT–PCR with specific primer pairs against mper2, or mrpl32 as a normalizing control. All results are expressed as the mean ± SEM of three independent experiments.