Selection of reference genes for gene expression studies in human neutrophils by real-time PCR

Initial screening of the expression pattern demonstrated that 3 of the 10 genes were expressed at very low levels in neutrophils and were excluded from further analysis. The range of expression stability of the other 7 genes was (from most stable to least stable): GNB2L1 (Guanine nucleotide binding protein, beta polypeptide 2-like 1), HPRT1 (Hypoxanthine phosphoribosyl transferase 1), RPL32 (ribosomal protein L32), ACTB (beta-actin), B2M (beta-2-microglobulin), GAPD (glyceraldehyde-3-phosphate dehydrogenase) and TBP (TATA-binding protein). Relative expression levels of the genes (from high to low) were: B2M, ACTB, GAPD, RPL32, GNB2L1, TBP, and HPRT1.Our data suggest that GNB2L1, HPRT1, RPL32, ACTB, and B2M may be suitable reference genes in gene expression studies of neutrophils.Neutrophils are the most numerous granulocytes in blood and are responsible for the first line of host defence. However, neutrophils have frequently been implicated in the pathogenesis of many diseases because they can produce various cytokines, chemokines and other proinflammatory mediators [1,2]. Numerous studies have been performed on the mechanisms that regulate the bioactivity of neutrophils. Understanding patterns of expressed genes may provide insight into complex regulatory networks and help to identify genes implicated in diseases. Quantitative real time PCR is one of the most powerful quantification methods for gene expression analysis. Similar to other methods used in expression studies, data from samples are usually required to be normalized against a set of data or references to correct for the difference in the amount of starting materials. The genes used as references are often referred to as housekeeping genes, assuming that those genes are constitutively expressed in certain tissues and under certain circumstances. However, the literature shows that the expression levels of the so called "housekeeping genes" may vary in different tissues, different cell types, and different