Email updates

Keep up to date with the latest news and content from BMC Research Notes and BioMed Central.

Open Access Research article

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

Carola Ledderose12, Jens Heyn2, Elisabeth Limbeck2 and Simone Kreth2*

Author Affiliations

1 Department of Anesthesiology and Intensive Care Medicine, Clinical Faculty Mannheim, University of Heidelberg, Germany

2 Department of Anesthesiology, Ludwig-Maximilians-University Munich, Germany

For all author emails, please log on.

BMC Research Notes 2011, 4:427  doi:10.1186/1756-0500-4-427

Published: 20 October 2011

Abstract

Background

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.

Results

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. IPO8, RPL13A, TBP and SDHA were identified as suitable reference genes in T cells. TBP, ACTB and SDHA were stably expressed in neutrophils. TBP and SDHA 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 IL-2 and FIH expression in T cells.

Conclusions

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.