RNA was extracted from tissues and LβT2 cells using TRIzol reagent (15596018, Invitrogen, Waltham, MA, USA) following the manufacturer’s protocol. For primary pituitary cultures, RNA was extracted using the Total RNA Mini Kit (FA32808-PS, Geneaid) following the manufacturer’s protocol. For the assessment of Tgfbr3l expression in different tissues and in LβT2 cells, 1 μg of total RNA was reverse-transcribed. For all other experiments, 200 ng (tissue) or 100 ng (primary culture) of total RNA was reverse-transcribed.
RNA was reverse-transcribed using random hexamers (C1181, Promega) and Moloney murine leukemia virus reverse transcriptase (M1701, Promega). The resulting cDNA was used for qPCR analysis using EvaGreen (ABMMmix, Diamed, Missisauga, ON, Canada) and primers listed in table S2 on a Corbett Rotorgene 600 instrument (Corbett Life Science, Sydney, NSW, Australia). mRNA levels were determined using the 2−ΔΔCT method. Gene expression was normalized to ribosomal protein L19 (Rpl19). All primers were validated for efficiency and specificity.
RNA was reverse-transcribed using random hexamers (C1181, Promega) and Moloney murine leukemia virus reverse transcriptase (M1701, Promega). The resulting cDNA was used for qPCR analysis using EvaGreen (ABMMmix, Diamed, Missisauga, ON, Canada) and primers listed in table S2 on a Corbett Rotorgene 600 instrument (Corbett Life Science, Sydney, NSW, Australia). mRNA levels were determined using the 2−ΔΔCT method. Gene expression was normalized to ribosomal protein L19 (Rpl19). All primers were validated for efficiency and specificity.