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

TLDR

Housekeeping genes are routinely used to normalize mRNA levels, but their expression can vary across tissues and conditions, making careful selection essential for accurate gene expression studies. The study aimed to evaluate seven candidate housekeeping genes in isolated human reticulocytes to determine the most stable reference. Using a simple ΔCt method, the authors compared pairwise gene expression within each of 31 reticulocyte samples and ranked stability based on repeatability of ΔCt values. GAPDH emerged as the most stable housekeeping gene, while B2M was the least stable and should be avoided for reticulocyte expression studies.

Abstract

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.