Fig. 1. Blood-circulating substances in young and aged mice.

a Cell-free miRNAs (cf-miRNAs) circulating with blood in young and aged mice. RNA was isolated from the plasma of young (6-week-old) and aged (>23-month-old) C57BL/6J mice, and miRNAs (indicated) were examined by qRT-PCR. The data were analyzed by the delta–delta Ct method using the data of cel-miR-39 as an external control and normalized with the data obtained from young mice as 1. Data are shown as mean ± SEM (n = 5 mice; *p < 0.05 by Wilcoxon rank-sum test). b Myogenic differentiation with young and aged mouse serum. C2C12 cells were cultured for 24 h in following the differentiation media: DMEM supplemented with 5% young mouse serum (MS, 6w) or 5% aged mouse serum (MS, >23m), and DMEM supplemented with 2% house serum (HS) as a conventional differentiation medium. Total RNA was extracted from the cells and undifferentiated C2C12 cells (day 0) as a control. The expression of Myog as a myogenic differentiation marker was examined by qRT-PCR and analyzed by the delta–delta Ct method, using the data of Gapdh as an internal reference. The data were further normalized with the data of undifferentiated cells (day 0) as 1. Data are shown as mean ± SEM (n = 3 independent determinations). c IGF-I level in young and aged mouse blood. Plasma samples were prepared from the blood of young and aged mice, and plasma levels of IGF-I were measured by ELISA. Data are shown as mean ± SEM (n = 5 mice).