title: Selection and validation of reliable reference genes for RT-qPCR analysis in a large cohort of pituitary adenomas.
abstract: Real-time reverse transcription quantitative PCR (RT-qPCR) has become the method of choice for quantification of gene expression changes. The most important limitations of RT-qPCR are inappropriate data normalization and inconsistent data analyses. Pituitary adenomas are common tumours, and the appropriate interpretation of increasingly published data within this field is prevented by the lack of a proper selection and validation of stably expressed reference genes.
  
  
  
  To find and validate the optimal reference gene or gene combination for reliable RT-qPCR gene expression in both non-functioning (NFPA) and hormone secreting (GH and ACTH) pituitary adenomas.
  
  
  
  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). The data was analysed using three programs: geNorm (Qbase+), Normfinder and BestKeeper having different algorithms to identify the most stable reference gene or combination of reference genes. Three reference genes ALAS1, PSMC4 and GAPDH, were selected for further validation in a larger cohort of 223 adenomas (141 NFPA, 63 GH and 19 ACTH).
  
  
  
  In all adenomas, ALAS1 and PSMC4 were the most stable reference genes as estimated by geNorm and Normfinder, whereas Bestkeeper ranked RPLP0 and ACTB as the two most stable out of 10 carefully selected genes. The best gene combination was PSMC4 and ALAS1 (geNorm) or PSMC4 and GAPDH (Normfinder). The validation experiment (geNorm) showed that the most stable gene combinations were ALAS1 and GAPDH in NFPA, and PSMC4 and GAPDH in hormone secreting adenomas.
  
  
  
  Several of the reference genes expressed good stability yielding several candidate genes. PSMC4 and ALAS1 were overall the most stably expressed genes in pituitary adenoma merely differing in ranking order. PSMC4 and ALAS1 have so far not been reported as reference genes in pituitary adenomas. The various reference gene algorithms showed a mixed selection of top ranked genes, thus suggesting a need for an individualised and rational choice of reference genes.
authors: Section of Specialized Endocrinology, Department of Endocrinology, Oslo University Hospital, Oslo, Norway; Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway; Research Institute for Internal Medicine, Oslo University Hospital, Oslo, Norway. Electronic address: normann.kjersti@medisin.uio.no.
authors: Kjersti Ringvoll
authors: KR
authors: Normann
authors: Section of Specialized Endocrinology, Department of Endocrinology, Oslo University Hospital, Oslo, Norway; Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
authors: Kristin Astrid Berland
authors: KAB
authors: Øystese
authors: Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway; Department of Medical Biochemistry, Oslo University Hospital, Oslo, Norway.
authors: Jens Petter
authors: JP
authors: Berg
authors: Research Institute for Internal Medicine, Oslo University Hospital, Oslo, Norway.
authors: Tove
authors: T
authors: Lekva
authors: Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway; Department of Neurosurgery, Oslo University Hospital, Oslo, Norway.
authors: Jon
authors: J
authors: Berg-Johnsen
authors: Section of Specialized Endocrinology, Department of Endocrinology, Oslo University Hospital, Oslo, Norway; Institute of Clinical Medicine, Faculty of Medicine, University of Oslo, Oslo, Norway.
authors: Jens
authors: J
authors: Bollerslev
authors: Section of Specialized Endocrinology, Department of Endocrinology, Oslo University Hospital, Oslo, Norway; Research Institute for Internal Medicine, Oslo University Hospital, Oslo, Norway.
authors: Nicoleta Cristina
authors: NC
authors: Olarescu
mesh_terms: D000236:Adenoma / Q000235:genetics*; D015331:Cohort Studies; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D010911:Pituitary Neoplasms / Q000235:genetics*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods* / Q000592:standards*; D012015:Reference Standards; D015203:Reproducibility of Results; D012984:Software
keywords: BestKeeper; Gene expression; MIQE guidelines; Non-functioning pituitary adenoma (NFPA); NormFinder; PCR array; geNorm