RNA was extracted from the arcuate nucleus of the hypothalamus of three male and three female mice at postnatal days 10, 12, 15, 18, 22, 26, 30, and 45. Pubertal development occurred between days 15 and 30 in these mice, as indicated by the increase in the expression of hypothalamic Tac2 (or neurokinin B) messenger RNA (mRNA).24 RNA was reverse-transcribed, and quantitative real-time PCR analysis was then performed to measure Mkrn3 mRNA levels, normalized to ribosomal protein L19 (see the Supplementary Appendix).
