Diurnal regulation of Rgs16 mRNA in liver and SCN. (A)Northern blot analysis of liver total RNA (20 μg/lane) isolated from individual female mice sacrificed at the indicated times (ZT, Zeitgeber Time; 12 hr light phase, ZT0-12; 12 hr dark phase, ZT12-24). Two mice collected at each time point were pair-caged, food and water ad libitum (representative of >8 mice/time point). Fold change (Δ) in Rgs16 mRNA levels are relative to basal expression at ZT16 (assayed by densitometry). To confirm equal loading in all lanes, the filter was rehybridized with a radiolabeled oligonucleotide complementary to 18s rRNA. (B) In situ hybridization of Rgs16 mRNA expression in SCN of ad libitum fed mice, 12 hr L:D. Fold change (Δ) in Rgs16 mRNA levels are relative to basal expression at ZT10 and ZT14. Relative Rgs16 mRNA levels were determined by densitometry of multiple sections obtained from each of two mice assayed at each time point. (C; left)Rgs16 mRNA expression (assayed by isotopic in situ hybridization) in coronal hemisections of SCN. Ad libitum fed mice were housed in constant darkness (CD) for two days prior to assay on day 3 at 6 hr into the presumptive light phase (CD6) and 2 hr into the presumptive dark phase (CD14). (C; right)Relative Rgs16 mRNA levels in SCN determined by densitometry of 2 sections from each of 4 mice per time point. Expression levels at CD6 and CD14 in Fig. 1C are similar to ZT6 and ZT14 in Fig. 1B, as expected of light entrained gene expression in SCN.