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Open Access Highly Accessed Methodology article

Selection of housekeeping genes for gene expression studies in human reticulocytes using real-time PCR

Nicholas Silver, Steve Best, Jie Jiang and Swee Lay Thein*

Author Affiliations

Molecular Haematology, Division of Gene and Cell Based Therapy, King's College London School of Medicine at King's College Hospital, Denmark Hill, London, SE5 9PJ, UK

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BMC Molecular Biology 2006, 7:33  doi:10.1186/1471-2199-7-33

Published: 6 October 2006

Abstract

Background

Control genes, which are often referred to as housekeeping genes, are frequently used to normalise 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 housekeeping genes is critical for gene expression studies. To address this issue, 7 candidate housekeeping genes including several commonly used ones were investigated in isolated human reticulocytes. For this, a simple ΔCt approach was employed by comparing relative expression of 'pairs of genes' within each sample. On this basis, stability of the candidate housekeeping genes was ranked according to repeatability of the gene expression differences among 31 samples.

Results

Initial screening of the expression pattern demonstrated that 1 of the 7 genes was expressed at very low levels in reticulocytes and was excluded from further analysis. The range of expression stability of the other 6 genes was (from most stable to least stable): GAPDH (glyceraldehyde 3-phosphate dehydrogenase), SDHA (succinate dehydrogenase), HPRT1 (hypoxanthine phosphoribosyl transferase 1), HBS1L (HBS1-like protein) and AHSP (alpha haemoglobin stabilising protein), followed by B2M (beta-2-microglobulin).

Conclusion

Using this simple approach, GAPDH was found to be the most suitable housekeeping gene for expression studies in reticulocytes while the commonly used B2M should be avoided.