Selection of reliable reference genes for quantitative real-time PCR in human T cells and neutrophils

• Main Authors: Ledderose Carola, Heyn Jens, Limbeck Elisabeth, Kreth Simone
• Format: Article
• Language: English
• Published: BMC 2011-10-01
• Series: BMC Research Notes
• Online Access: http://www.biomedcentral.com/1756-0500/4/427

<p>Abstract</p> <p>Background</p> <p>The choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR (qPCR). This method is frequently applied to study gene expression patterns in immune cells, yet a thorough validation of potential reference genes is still lacking for most leukocyte subtypes and most models of their in vitro stimulation. In the current study, we evaluated the expression stability of common reference genes in two widely used cell culture models-anti-CD3/CD28 activated T cells and lipopolysaccharide stimulated neutrophils-as well as in unselected untreated leukocytes.</p> <p>Results</p> <p>The mRNA expression of 17 (T cells), 7 (neutrophils) or 8 (unselected leukocytes) potential reference genes was quantified by reverse transcription qPCR, and a ranking of the preselected candidate genes according to their expression stability was calculated using the programs NormFinder, geNorm and BestKeeper. <it>IPO8</it>, <it>RPL13A</it>, <it>TBP </it>and <it>SDHA </it>were identified as suitable reference genes in T cells. <it>TBP</it>, <it>ACTB </it>and <it>SDHA </it>were stably expressed in neutrophils. <it>TBP </it>and <it>SDHA </it>were also the most stable genes in untreated total blood leukocytes. The critical impact of reference gene selection on the estimated target gene expression is demonstrated for <it>IL-2 </it>and <it>FIH </it>expression in T cells.</p> <p>Conclusions</p> <p>The study provides a shortlist of suitable reference genes for normalization of gene expression data in unstimulated and stimulated T cells, unstimulated and stimulated neutrophils and in unselected leukocytes.</p>