Abstract
Gene expression studies employing real-time PCR has become an intrinsic part of biomedical research. Appropriate normalization of target gene transcript(s) based on stably expressed housekeeping genes is crucial in individual experimental conditions to obtain accurate results. In multiple sclerosis (MS), several gene expression studies have been undertaken, however, the suitability of housekeeping genes to express stably in this disease is not yet explored. Recent research suggests that their expression level may vary under different experimental conditions. Hence it is indispensible to evaluate their expression stability to accurately normalize target gene transcripts. The present study aims to evaluate the expression stability of seven housekeeping genes in rat granule neurons treated with cerebrospinal fluid of MS patients. The selected reference genes were quantified by real time PCR and their expression stability was assessed using GeNorm and NormFinder algorithms. GeNorm identified transferrin receptor (Tfrc) and microglobulin beta-2 (B2m) the most stable genes followed by ribosomal protein L19 (Rpl19) whereas β-actin (ActB) and glyceraldehyde-3-phosphate-dehydrogenase (Gapdh) the most fluctuated ones in these neurons. NormFinder identified Tfrc as the best invariable gene followed by B2m and Rpl19. ActB and Gapdh were the least stable genes as analyzed by NormFinder algorithm. Both methods reported Tfrc and B2m the most stably expressed genes and Gapdh the least stable one. Altogether our data demonstrate the significance of pre-validation of housekeeping genes for accurate normalization and indicates Tfrc and B2m as best endogenous controls in MS. ActB and Gapdh are not recommended in gene expression studies related to current one.
Highlights
Techniques employed for calibrating gene expression are paramount in studies directed toward accurate analysis of transcriptomic profiles
The conventional way to perform normalization is to select a housekeeping gene whose expression is believed to remain stable in all cell types/tissues, during cellular development and under various experimental conditions relate the expression of gene of interest to that of a housekeeping gene
GADPH, on the other hand, is a key glycolytic enzyme involved mainly in the production of energy. Since both ActB and Gapdh are involved in maintaining the basic metabolic functions of a cell, they are presumed to express at stable levels
Summary
Techniques employed for calibrating gene expression are paramount in studies directed toward accurate analysis of transcriptomic profiles. The expression of target gene transcripts is normalized with an internal control, often referred to as a housekeeping gene. Housekeeping (HK) genes are endogenous controls that are required for the primary function of a cell their expression should be constant in all conditions. Invariable expression of the so-called housekeeping genes has been observed during cellular development (Al-Bader and Al-Sarraf, 2005) and under distinct experimental conditions (Zhong and Simons, 1999; Hamalainen et al, 2001; Deindl et al, 2002; Glare et al, 2002; Torres et al, 2003; Radonic et al, 2004; Toegel et al, 2007; Gubern et al, 2009). It is recommended that more than one stably expressed gene should be used for precise normalization procedure (Zhong and Simons, 1999; Tricarico et al, 2002; Vandesompele et al, 2002; Ohl et al, 2005)
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