Abstract
BackgroundReference genes, which are often referred to housekeeping genes, are frequently used to normalize mRNA levels between different samples. However the expression level of these genes may vary among tissues or cells, and may change under certain circumstances. Thus the selection of reference gene(s) is critical for gene expression studies. For this purpose, 10 commonly used housekeeping genes were investigated in isolated human neutrophils.ResultsInitial 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.ConclusionOur data suggest that GNB2L1, HPRT1, RPL32, ACTB, and B2M may be suitable reference genes in gene expression studies of neutrophils.
Highlights
Reference genes, which are often referred to housekeeping genes, are frequently used to normalize mRNA levels between different samples
The principle that the expression ratio of two ideal reference genes should be identical in all samples is well established. Based on this principle we found GNB2L1, Hypoxanthine phosphoribosyltransferase 1 (HPRT1), ribosomal protein L32 (RPL32), ACTB, and B2M were stably expressed in the neutrophils, and they were used for the calculation of a normalization factor (NF)
We found GNB2L1, HPRT1, RPL32, ACTB, and B2M are good choices for reference gene(s) selection
Summary
Reference genes, which are often referred to housekeeping genes, are frequently used to normalize mRNA levels between different samples. The expression level of these genes may vary among tissues or cells, and may change under certain circumstances. The selection of reference gene(s) is critical for gene expression studies. For this purpose, 10 commonly used housekeeping genes were investigated in isolated human neutrophils. The literature shows that the expression levels of the so called "housekeeping genes" may vary in different tissues, different cell types, and different disease stages [3,4,5,6].
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