Validation of reference genes for quantitative gene expression analysis in experimental epilepsy

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Epilepsy is a chronic neurological condition that is characterized by recurrent, unprovoked seizures (Thurman et al., 2011). It is one of the world's most common neurological disorders, affecting approximately 1% of the world population (∼65 million) with around 2.4 million new diagnoses annually (Thurman et al., 2011). Temporal lobe epilepsy (TLE), where seizures originate in the temporal lobe, is the most common epilepsy syndrome, often refractory to treatment, and is thought to be caused by a brain insult (Chang & Lowenstein, 2003). TLE is characterized by hippocampal atrophy and limited extrahippocampal damage as well as seizures that originate in the hippocampus and/or closely related structures (Spencer & Spencer, 1994). TLE is most commonly modeled in rats by one of three methods: electrical stimulation (Norwood et al., 2010; Sloviter & Damiano, 1981), systemic administration of kainic acid (Ben‐Ari & Lagowska, 1978; Ben‐Ari, Lagowska, Tremblay, & Le Gal La Salle, 1979), or systemic administration of pilocarpine (Turski, Cavalheiro, et al., 1983; Turski, Czuczwar, Kleinrok, & Turski, 1983).
Gene expression analysis is a standard approach for studying the regulation of biological mechanisms under normal and diseased conditions. A better understanding of the molecular mechanisms at play in epilepsy is crucial for the development of novel therapeutics that correct culpable dysfunction. Quantitative real‐time polymerase chain reaction (qPCR) is the gold standard for gene expression analysis in small quantities of tissue. Appropriate reference genes (formerly known as housekeeping genes) are required in order to precisely and accurately determine the expression of genes of interest (GOIs) (Bustin et al., 2009). The purpose of normalizing data to one or more reference genes is to account for differences in the amount of cDNA (McCulloch, Ashwell, O'Nan, & Mente, 2012) and efficiency of amplification (Vandesompele et al., 2002), and to compare GOIs among different samples. Reference genes are typically involved in the basic maintenance of cellular structure and/or function. Irrespective of its role, reference gene mRNA should express at a constant level in all conditions, regardless of cell cycle stage or age (Eisenberg & Levanon, 2013; Radonić et al., 2004). Reference genes vary widely across diseases and experimental models (Bademci et al., 2010) but can be considered suitable if several criteria are met (Chervoneva et al., 2010), the most important criterion being stable expression. The expression of a reference gene cannot be influenced by experimental conditions (Kozera & Rapacz, 2013).
Reference genes have been proposed in a few studies on human and experimental epilepsy (Maurer‐Morelli et al., 2012; Pernot, Dorandeu, Beaup, & Peinnequin, 2010). There has, however, not been a systematic evaluation or validation of potential reference genes in any experimental epilepsy model. The aim of this study was to discover reference genes for two rat models of epilepsy: 8‐hr perforant pathway stimulation (PPS) (Norwood et al., 2010) and systemic kainate‐lorazepam (KaL) during epileptogenesis and/or chronic epilepsy, and after acute, noninjurious seizures (30‐min PPS) (Norwood et al., 2010). The mRNA expression levels of 15 (Kienzler‐Norwood et al., 2017) potential reference genes were determined in hippocampi from treated and control animals. Four different validated and established methods to determine expression stability were used: geNorm (Vandesompele et al., 2002), NormFinder (Andersen, Jensen, & Ørntoft, 2004), BestKeeper (Pfaffl, Tichopad, Prgomet, & Neuvians, 2004), and Delta‐Ct (ΔCt) (Silver, Best, Jiang, & Thein, 2006).

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