Abstract
BackgroundThe 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.ResultsThe 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.ConclusionsThe 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.
Highlights
The choice of reliable reference genes is a prerequisite for valid results when analyzing gene expression with real-time quantitative PCR
When selecting potential reference genes for the cell culture model of unstimulated and LPS-treated neutrophils (n = 7 paired samples), we focused on the set of seven pre-selected genes that we had evluated in T cells, with one slight modification: instead of HBB, which had been consistently ranked last in T cells by all three analyzing programs (Table 3), ACTB was included, as it is one of the most commonly used reference genes [6] and has previously been suggested for normalization of gene expression in untreated neutrophils [12]
We evaluated, to our knowledge for the first time, the expression stability of common reference genes separately in two widely-used cell culture models of stimulated leukocyte subtypes: T cells activated by antiCD3/CD28 beads, and LPS-stimulated neutrophils
Summary
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. The most commonly applied normalization strategy involves the use of reference genes as internal controls, whose expression should be constant in all samples under investigation [7] Since it has become clear, though, that conventional reference genes, such as glyceraldehyde-3-. Stimulating T cells with anti-CD3/CD28 beads to mimic the activation by antigen-presenting cells [13], for example, or treating neutrophils with lipopolysaccharide (LPS) [14,15,16] are two well-established in vitro models in the investigation of inflammatory, infectious or autoimmune disease; a systematic validation of reference gene stability has far been lacking for either model, though
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