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. 2005 Feb 18:6:4.
doi: 10.1186/1471-2199-6-4.

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

Xiaozhu Zhang  1 , Lily Ding, Andrew J Sandford
Affiliations

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

Xiaozhu Zhang et al. BMC Mol Biol. .

Abstract

Background: Reference 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.

Results: 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.

Conclusion: Our data suggest that GNB2L1, HPRT1, RPL32, ACTB, and B2M may be suitable reference genes in gene expression studies of neutrophils.

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Figures

Figure 1
Figure 1
The results of RNA analysis by Agilent bioanalyzer. The first peak is a 20 bp molecular marker. The second and the third peaks are 18S and 28S rRNA.
Figure 2
Figure 2
Gene expression stability of seven candidate reference genes in the neutrophil analyzed by the geNorm program. The threshold for eliminating a gene as unstable was M ≥ 0.5.
Figure 3
Figure 3
The relative expression level normalized against Normalization Factors from the 5 most stable genes (HPRT1, GNB2L1, RPL32, ACTB, and B2M) provided by geNorm. HPRT1 was the lowest expressed gene, and B2M was the highest among the candidate genes in neutrophils.
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