RNA Extraction and RT-qPCR From Cultured Cells and Pituitaries of Knockout Mice

Frozen pituitaries were thawed and homogenized in TRIzol, and RNA was extracted following the manufacturer's protocol. Total RNA from pituitaries (200 ng) or LβT2b cells (500 ng) was reverse transcribed using Moloney murine leukemia virus reverse transcriptase and random hexamer primers. qPCR reactions were performed using Blastaq Master mix on a Corbett Rotorgene 6000 instrument (Corbett Life Science, Mortlake, NSW, Australia). For pituitary cDNA, expression of Rpl19, Fshb, Lhb, Cga, Gnrhr, Atf3, Jun, Junb, Fos, and Egr1 was analyzed in duplicate. For LβT2b cDNA, expression of Rpl19, Atf3, Fshb, and Junb was analyzed in triplicate. Gene expression was determined relative to that of the reference gene Rpl19 using the 2-ΔΔCt method. Primer sets are listed in Table 1.