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

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 DeltaCt 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.

Figure 1

Q-RT-PCR cycle threshold values for 7 candidate housekeeping genes among 31 adult human reticulocyte samples. Candidate housekeeping genes include AHSP, B2M, GAPDH, HBS1L, HPRT1 and SDHA.

Figure 2

ΔCt approach to housekeeping gene selection. ΔCt variability in candidate housekeeping gene comparisons are shown as medians (lines), 25^th percentile to the 75^th percentile (boxes) and ranges (whiskers) for 31 human reticulocyte samples. A – highlights a subset of candidate housekeeping genes including B2M, GAPDH and SDHA. B – shows comparisons of the complete set of candidate housekeeping genes, which include AHSP, B2M, GAPDH, HBS1L, HPRT1 and SDHA.

Figure 3

Housekeeping validation: The above plots show the relationship between comparative Ct for γ-globin normalised to a) B2M, b) SDHA and c) GAPDH and Hb F. The ideal scenario is for the γ-globin vs Hb F correlation to improve as progressively better housekeeping genes are used.