... second-roundPCRs consisted of five cycles for 30 s at 94 °C, 30 s at45 °C and 2.5 min at 72 °C, and of 30 cycles for 30 s at94 °C, 30 s at 50 °C and 2.5 min at 72 °C(5.5minforlastcycle). The second-round ... complementary to nucle-otides 416–435). Both first-round and second-round PCRswere performed for 35 cycles for 30 s at 94 °C, 30 s at 55 °C and 1 min at 72 °C (11 min for last cycle). The second-round ... hypothalamic RFamide-related peptide identified byimmunoaffinity chromatography and mass spectrometry. FEBSLett. 512, 255–258.15. Fukusumi, S., Habata, Y. , Yoshida, H., Iijima, N., Kawamata, Y. ,...