Proceedings of The Physiological Society

Europhysiology 2018 (London, UK) (2018) Proc Physiol Soc 41, PCA298

Poster Communications

Selection of effective reference genes for expression studies: RFRP-3 and GPR147 signaling in rat hippocampus

S. Kutlu1,2, E. Kurar1,3, C. Eroglu1,3, A. Aydin1,4, H. H. Kozak1,5, Z. Sahin6

1. Necmettin Erbakan University Neuroscience Research and Application Center (NENRC), Konya, Turkey. 2. Department of Physiology, Necmettin Erbakan University Meram Faculty of Medicine, Konya, Turkey. 3. Department of Medical Biology, Necmettin Erbakan University Meram Faculty of Medicine, Konya, Turkey. 4. Department of Psychiatry, Necmettin Erbakan University Meram Faculty of Medicine, Konya, Turkey. 5. Department of Neurology, Necmettin Erbakan University Meram Faculty of Medicine, Konya, Turkey. 6. Department of Physiology, Karadeniz Technical University, Faculty of Medicine, Trabzon, Turkey.


Real time-polymerase chain reaction (RT-PCR) is widely used for investigation of gene function and interactions at mRNA level in part of functional genomics studies. Numerous factors including sample to PCR profile cause variation and misevaluation of expression levels in quantitative RT-PCR (qPCR) analyses. Housekeeping genes (HKG), that are assumed to be constantly expressed in all cells and tissues, can be thereby used for as an internal control and normalization of variations between samples and analyses. Although several HKGs were available for this purpose, recent research indicated that expressions of some HKGs are biologically affected from various experimental applications, which may cause wrong normalization and evaluation of results. Objective of this study was to select appropriate HKGs to be used in the normalization of gene expression of RFamide related peptide-3 (RFRP-3) and its receptor (GPR147) signaling on anxiety like behaviors and neurogenesis in rat hippocampus. Osmotic brain minipumps were implanted to 24 rats (male, 300-350 g) which were divided into four groups including control (n=6), RFRP-3 (1 nMol/day, n=6), RF9 (RFRP-3 antagonist; 10 nMol/day, n=6) and RFRP-3 + RF9 (n=6). Drug infusions into lateral ventricles were performed for 15 days. Total RNA isolation and cDNA synthesis were performed from hippocampus tissues. Expressions of 9 HKGs (GAPDH, SDHA, RPL13A, PGK1, HPRT1, 18SrRNA, ACTB, YWHAZ and CycA) at mRNA levels were evaluated using qPCR. Average expression stability values (M) were separately calculated for each groups using BestKeeper, geNorm and NormFinder software. Both results of geNorm and Normfinder algorithms indicated that PGK1, YWHAZ and GAPDH were the most stable genes, respectively. As a conclusion, PGK1 and YWHAZ seems to be the most suitable reference genes for studying RFRP-3 and GPR147 signaling on anxiety like behaviors and neurogenesis in rat hippocampus.

Where applicable, experiments conform with Society ethical requirements