Endocrine Abstracts

Background: Real-time reverse transcription quantitative PCR (RT-qPCR) has become the method of choice for quantification of gene expression changes. Inappropriate data normalization and inconsistent data analyses are some limitations of RT-qPCR. Pituitary adenomas are frequent tumours and the interpretation of increasingly published data within this field is hindered by the lack of a proper selection and validation of stably expressed reference genes.

Aim: To find and validate the optimal reference gene or combination for reliable RT-qPCR gene expression in both non-functioning (NFPA) and hormone secreting (ACTH and GH) pituitary adenomas.

Material and methods: Thirty commonly used reference genes (PCR array reference gene panel, BioRad, Hercules, CA) were quantified by RT-qPCR in 24 pituitary adenomas (12 NFPA, 8 GH and 4 ACTH). Data were analyzed using three programs: GeNorm (Qbase+), Normfinder and BestKeeper having different algorithms to identify the most stable reference gene/combination of reference genes. The top candidate genes were validated in a larger cohort of adenomas (144 NFPA, 63 GH and 19 ACTH).

Results: In all adenomas, ALAS1 and PSMC4 were the most stable reference genes as estimated by GeNorm and Normfinder, whereas Bestkeeper ranked RPL30 and RPS17 as the two most stable genes out of 10 carefully selected genes. The best gene combination was ALAS1 and PSMC4 (GeNorm) or PSMC4 and GAPDH (Normfinder). The validation experiment (GeNorm) showed that ALAS1 and PSMC4 in NFPA and PSMC4 and GAPDH in hormone secreting adenomas were the most stable gene combination. The best ranked reference genes were not identical in different subgroups of adenomas.

Conclusions: Differences in stability between top-ranked genes in the reference gene selection programs were minor. PSMC4, ALAS1 and GAPDH were stably expressed in pituitary adenomas and a combination of two of them will serve as the most robust choice of reference genes.