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Title: Selection of reference genes for gene expression studies in rat oligodendrocytes using quantitative real time PCR
Authors: NELISSEN, Katherine 
SMEETS, Karen 
Mulder, M.
AMELOOT, Marcel 
Issue Date: 2010
Publisher: Elsevier
Abstract: Quantitative real time polymerase chain reaction (qPCR) has become a widely used tool to examine gene expression levels. Reliable quantification, however, depends on a proper normalization strategy. Normalization with multiple reference genes is becoming the standard, although the most suitable reference genes depend on the applied treatment as well as the tissue or cell type studied. In this study the stability of various reference genes was investigated in cultures of oligodendrocytes derived from either mature or neonatal rats, the latter also in the presence of the liver X receptor (LXR) agonist. The expression stability of ten commonly used reference genes (HPRT, GAPDH, 18S, ActB, CycA, Tbp, Rpl13A, YWHAZ, HMBS, Pgk1) was analyzed using geNorm and NormFinder. When comparing the different types of cell cultures, Rpl13A, CycA, Pgk1 and YWHAZ were identified as most stable genes. After LXR agonist treatment, CycA, Pgk1 and Rpl13A were found to be the most stable by both geNorm and NormFinder. HMBS and the commonly used housekeeping genes GAPDH and 18S turned out to be the most variable according to geNorm and NormFinder. In conclusion, the use of multiple reference genes, instead of only one, in qPCR experiments with rat oligodendrocytes is strongly advised and standard housekeeping genes such as GAPDH and 18S are not recommended as they appear to be relatively unstable under the experimental conditions used. Reference gene selection should always be performed for each individual experiment, since useful reference genes are very specific for every situation.
Notes: [Nelissen, Katherine; Ameloot, Marcel] Hasselt Univ, Biomed Res Inst, Lab Cell Physiol, B-3590 Diepenbeek, Belgium - [Nelissen, Katherine; Hendriks, Jerome J. A.; Ameloot, Marcel] Transnat Univ Limburg, B-3590 Diepenbeek, Belgium. [Smeets, Karen] Hasselt Univ, Ctr Environm Sci, B-3590 Diepenbeek, Belgium. [Mulder, Monique] Rotterdam Univ, Div Pharmacol Vasc & Metab Dis, Dept Internal Med, NL-3015 CE Rotterdam, Netherlands. [Hendriks, Jerome J. A.] Hasselt Univ, Biomed Res Inst, Immunol Lab, B-3590 Diepenbeek, Belgium.
Keywords: Glial cells,internal control gene, normalization, Genorm NormFinder;biochemical research methods; neurosciences
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ISSN: 0165-0270
e-ISSN: 1872-678X
DOI: 10.1016/j.jneumeth.2009.12.018
ISI #: 000275769900012
Category: A1
Type: Journal Contribution
Validations: ecoom 2011
Appears in Collections:Research publications

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