AUTHOR=Mathur Deepali , Lopez-Rodas Gerardo , Ureña-Peralta Juan , Casanova Bonaventura , Coret-Ferrer Francisco , Burgal-Marti Maria 

TITLE=Bypassing hazard of housekeeping genes: their evaluation in rat granule neurons treated with cerebrospinal fluid of multiple sclerosis subjects

JOURNAL=Frontiers in Cellular Neuroscience

VOLUME=Volume 9 - 2015

YEAR=2015

URL=https://www.frontiersin.org/journals/cellular-neuroscience/articles/10.3389/fncel.2015.00375

DOI=10.3389/fncel.2015.00375

ISSN=1662-5102

ABSTRACT=<p>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 <italic>GeNorm</italic> and <italic>NormFinder</italic> algorithms. <italic>GeNorm</italic> identified transferrin receptor (<italic>Tfrc</italic>) and microglobulin beta-2 (<italic>B2m</italic>) the most stable genes followed by ribosomal protein L19 (<italic>Rpl19</italic>) whereas β-actin (<italic>ActB</italic>) and glyceraldehyde-3-phosphate-dehydrogenase (<italic>Gapdh</italic>) the most fluctuated ones in these neurons. <italic>NormFinder</italic> identified <italic>Tfrc</italic> as the best invariable gene followed by <italic>B2m</italic> and <italic>Rpl19</italic>. <italic>ActB</italic> and <italic>Gapdh</italic> were the least stable genes as analyzed by <italic>NormFinder</italic> algorithm. Both methods reported <italic>Tfrc</italic> and <italic>B2m</italic> the most stably expressed genes and <italic>Gapdh</italic> the least stable one. Altogether our data demonstrate the significance of pre-validation of housekeeping genes for accurate normalization and indicates <italic>Tfrc</italic> and <italic>B2m</italic> as best endogenous controls in MS. <italic>ActB</italic> and <italic>Gapdh</italic> are not recommended in gene expression studies related to current one.</p>