Determination of Appropriate Endogenous Reference Genes for RT-qPCR Analysis in Syrian (Golden) Hamsters and Mongolian Gerbils

Year 2024, Volume: 8 Issue: 3, 533 - 542, 30.09.2024

Bülent Gündüz , Betül Önder , Tanay Uzgan

https://doi.org/10.30621/jbachs.1315023

Abstract

Purpose: The use of hamsters and gerbils has increased significantly in a variety of fields, including
biological rhythms, reproductive biology, immunology, oncology, and many others.
Material and Methods: The most stable genes in Syrian hamsters (Mesocricetus auratus) and
Mongolian gerbils (Meriones unguiculatus) were assessed using 32 reference genes for normalization
in RT-qPCR analysis. Adrenal, cerebral cortex, heart, hypothalamus, kidney, liver, lung and testis
tissues were used to extract and purify RNAs. GeNorm was used to determine the gene expression
stabilities of 14 candidate endogenous genes from each tissue that was compatible for both animals.

Results: Under our experimental conditions, we discovered that two endogenous genes are adequate
for each tissue to perform RT-qPCR normalization. There were differences in the most stable genes
between species and tissues.

Conclusion: We suggest that combinations of endogenous genes ought to be carefully chosen under
various experimental circumstances.

Keywords

hamsters, gerbils, reference genes, endogenous genes, qPCR

Supporting Institution

TÜBİTAK COST (European Cooperation in Science and Technology COST Association)

Project Number

Grant No: 111T639, 2011

Thanks

This research was supported by TÜBİTAK COST (European Cooperation in Science and Technology COST Association) (Grant No: 111T639, 2011)

Search for the Correct Endogenous Reference Genes by RT-qPCR in Syrian (Golden) Hamsters and Mongolian Gerbils

Abstract

Project Number

Grant No: 111T639, 2011

References

• Bustin SA. Real-time, fluorescence-based quantitative PCR: A snapshot of current procedures and preferences. Expert Rev Mol Diagn 2005; 5: 493-498.
• Bustin SA, Nolan T. Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction. J Biomol Tech 2004; 15: 155.
• Chapman JR, Waldenström J. With reference to reference genes: A systematic review of endogenous controls in gene expression studies. PLoS One 2015; 10: e0141853.
• Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M, et al. The MIQE guidelines: Minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009; 55: 611–622.
• Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods 2001;25: 402–408.
• Kanakachari M, Solanke AU, Prabhakaran N, Ahmad I, Dhandapani G, Jayabalan N, et al. Evaluation of suitable reference genes for normalization of qpcr gene expression studies in Brinjal (Solanum melongena L.) during fruit developmental stages. Appl Biochem Biotechnol 2016; 178: 433–450.
• Kouadjo KE, Nishida Y, Cadrin-Girard JF, Yoshioka M, St-Amand J. Housekeeping and tissue-specific genes in mouse tissues. BMC Genom 2007; 8: 1–16.
• Silver N, Best S, Jiang J, Thein SL. Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR. BMC Mol Biol 2006; 7: 1–9.
• Andersen CL, Jensen JL, Ørntoft TF. Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res 2004; 64: 5245–5250.
• Vandesompele J, de Preter K, Pattyn F, Poppe B, van Roy N, de Paepe A, et al. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002; 3: 1–12.
• Gündüz B, Stetson MH Maternal transfer of photoperiodic information in Siberian hamsters. vi. effects of time-dependent 1-hr melatonin infusions in the mother on photoperiod-induced testicular development of her offspring. J Pineal Res 2003;34: 217–225.
• Mason AO, Duffy S, Zhao S, Ubuka T, Bentley GE, Tsutsui K, et al. Photoperiod and reproductive condition are associated with changes in rfamide-related peptide (RFRP) expression in Syrian hamsters (Mesocricetus auratus). J Biol Rhythms 2010; 25:176-185.http://doi.org/10.1177/0748730410368821 25, 176–185.
• Reiter RJ, Li K, Gonzalez‐Brito, Tannenbaum MG, Vaughan MK, Vaughan GM, et al. Elevated environmental temperature alters the responses of the reproductive and thyroid axes of female Syrian hamsters to afternoon melatonin injections. J Pineal Res 1988; 5: 301–315.
• Tournier BB, Menet JS, Dardente H, Poirel VJ, Malan A, Masson-Pévet M, et al. Photoperiod differentially regulates clock genes’ expression in the suprachiasmatic nucleus of Syrian hamster. Neuroscience 2003; 118: 317–322.
• Henningsen JB, Ancel C, Mikkelsen JD, Gauer F, Simonneaux V. Roles of RFRP-3 in the daily and seasonal regulation of reproductive activity in female Syrian hamsters. Endocrinology 2017; 158: 652–663.
• Hirose M, Ogura A. The golden (Syrian) hamster as a model for the study of reproductive biology: Past, present, and future. Reprod Med Biol 2019; 18: 34–39.
• Beery AK, Paul MJ, Routman DM, Zucker I. Maternal photoperiodic history affects offspring development in Syrian hamsters. J Biol Rhythms 2008; 23: 445–455.
• Gündüz B, Okimoto DK. Methyl donor supplementation alters serum leptin levels and increases appetite but not body weight in cross-fostered male Syrian hamster offspring (Mesocricetus auratus). J Anim Physiol Anim Nutr (Berl) 2022; 106: 1130–1138.
• Gündüz B, Stetson MH. A test of the coincidence and duration models of melatonin action in Siberian hamsters: the effects of 1-hr melatonin infusions on testicular development in intact and pinealectomized prepubertal Phodopus sungorus. J Pineal Res 2001; 30: 97–107.
• Schulz KM, Sisk CL. Pubertal hormones, the adolescent brain, and the maturation of social behaviors: Lessons from the Syrian hamster. Mol Cell Endocrinol 2006; 254: 120–126.
• Miao J, Chard LS, Wang Z, Wang Y. Syrian hamster as an animal model for the study on infectious diseases. Front Immunol 2019; 10:2329.
• Rosenke K, Meade-White K, Letko M, Clancy C, Hansen F, Liu Y, et al. Defining the Syrian hamster as a highly susceptible preclinical model for SARS-CoV-2 infection. Emerg Microbes Infect 2020; 9: 2673–2684.
• Thomas MA, Spencer JF, la Regina MC, Dhar D, Tollefson AE, Toth K, et al. Syrian hamster as a permissive immunocompetent animal model for the study of oncolytic adenovirus vectors. Cancer Res 2006; 66: 1270–1276.
• Karakaş A, Çamsari Ç, Serin E, Gündüz B. Effects of photoperiod and food availability on growth, leptin, sexual maturation and maintenance in the Mongolian gerbils (Meriones unguiculatus). Zool Sci 2005; 22: 665–670.
• Saltzman W, Ahmed S, Fahimi A, Wittwer DJ, Wegner FH. Social suppression of female reproductive maturation and infanticidal behavior in cooperatively breeding Mongolian gerbils. Horm Behav 2006; 49: 527–537.
• Hu H, Kang C, Hou X, Zhang Q, Meng Q, Jiang J, et al. Blue light deprivation produces depression-like responses in Mongolian gerbils. Front Psychiatry 2020; 11: 233.
• Romero-Morales L, García-Saucedo B, Martínez-Torres M, et al. Paternal and infanticidal behavior in the Mongolian gerbil (Meriones unguiculatus): An approach to neuroendocrine regulation. Behav Brain Res 2021; 415: 113520.
• Yamaguchi H, Kikusui T, Takeuchi Y, Yoshimura H, Mori Y. Social stress decreases marking behavior independently of testosterone in Mongolian gerbils. Horm Behav 2005; 47: 549–555.
• Dehmel S, Kopp-Scheinpflug C, Dörrscheidt GJ, Rübsamen R. Electrophysiological characterization of the superior paraolivary nucleus in the Mongolian gerbil. Hear Res 2002; 172:18–36.
• Laumen G, Tollin DJ, Beutelmann R, Klump GM. Aging effects on the binaural interaction component of the auditory brainstem response in the Mongolian gerbil: Effects of interaural time and level differences. Hear Res 2016; 337: 46–58.
• Bleich EM, Martin M, Bleich A, Klos A. The Mongolian gerbil as a model for inflammatory bowel disease. Int J Exp Pathol 2010; 91: 281–287.
• Noto JM, Romero-Gallo J, Blanca Piazuelo M, Peek RM. The Mongolian gerbil: A robust model of helicobacter pylori-induced gastric inflammation and cancer. Methods Mol Biol 2016; 1422: 263–280.
• Elliott JA. Circadian rhythms and photoperiodic time measurement in mammals. Fed. Proc 1976; 35, 2339–2346.
• Hoffmann K. The critical photoperiod in the Djungarian hamster Phodopus sungorus. In Vertebrate circadian systems, Berlin: Springer; 1982: pp. 297-304.
• Karakaş A, Gündüz B. Effect of different photoperiods on gonadal maintenance and development in Mongolian gerbils (Meriones unguiculatus). Zool Sci 2002;19: 233-239.
• Benimettskii Y. Seasonal changes in the relative weight of the adrenals and gonads in the Mongolian gerbil. Ekologiya 1975; 2:95–96.
• Darrow JM, Davis FC, Elliott JA, Stetson MH, Turek FW, Menaker M. Influence of photoperiod on reproductive development in the Golden hamster. Biol Reprod 1980; 22: 443–450.
• Gaston S, Menaker M. Photoperiodic control of hamster testis. Science 1967;158, 925–928.
• Horton T. Cross-fostering of voles demonstrates in utero effect of photoperiod and the pineal gland. Biol Reprod 1985;41, 924–939.
• Horton TH. Growth and reproductive development of male Microtus montanus is affected by the prenatal photoperiod. Biol Reprod 1984;31: 499–504.
• Lee TM, Zucker I. Vole infant development is influenced perinatally by maternal photoperiodic history. Am J Physiol Regul Integr Comp Physiol 1988;255: R831-R838.
• Rollag MD, Dipinto MN, Stetson MH. Ontogeny of the gonadal response of Golden hamsters to short photoperiod, blinding, and melatonin. Biol Reprod 1982; 27: 898–902.
• Stetson MH, Elliott JA, Goldman BD. Maternal transfer of photoperiodic information influences the photoperiodic response of prepubertal Djungarian hamsters (Phodopus sungorus sungorus). Biol Reprod 1986; 34: 664–669.
• Li B, Matter EK, Hoppert HT, Grayson BE, Seeley RJ, Sandoval DA. Identification of optimal reference genes for RT-qPCR in the rat hypothalamus and intestine for the study of obesity. Int J Obes 2014; 38:192–197.
• Rueda-Martínez C, Fernández MC, Soto-Navarrete MT, Jiménez-Navarro M, Durán AC, Fernández B. Identification of reference genes for quantitative real time pcr assays in aortic tissue of Syrian hamsters with bicuspid aortic valve. PLoS One 2016;11:e0164070.
• Bahr SM, Borgschulte T, Kayser KJ, Lin N. Using microarray technology to select housekeeping genes in Chinese hamster ovary cells. Biotechnol Bioeng 2009;104:1041–1046.