Abstract
Background: Changes in gene expression are increasingly used to evaluate the effects of exposure to environmental agents. Housekeeping genes (Hk) are essential in these analyzes as internal controls to normalize expression levels assessed in real-time PCR (RT-PCR). Ideal Hk genes (i) are constitutively expressed; (ii) do not respond to external stimuli and (iii) show small or no variation between samples or from one assay to another. Previous studies indicate that some commonly used Hk genes, including glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and beta-actin, have differential expression in various cell lines. The objective of this study is to identify and validate the most appropriate housekeeping genes for RNA expression analysis of human primary peripheral blood mononuclear cells (PBMCs) in response to antigen like stimulation. Methods: The expression of the five most commonly used genes for a genomic study of lymphocytes was determined [HPRT, 18S, GAPDH, Ubiquitin C (UBC), beta-2 microglobulin (B2M)] on PBMCs, isolated from fresh blood, following activation with anti-CD3 and anti-CD28. This leads to the activation and proliferation of T lymphocytes in order to modulate the expression of specific genes. Results : Using reverse transcriptase RT-PCR protocol, we show that following activation only B2M remain unchanged in PBMCs. This result has been confirmes In contrast, 18S, HPRT and GAPDH show significant variation in PBMC gene expression. Conclusion: Although our results suggest that the relevance of Hk genes should be determined for each experimental condition, B2M appear to be excellent candidate as internal controls. Keywords : Housekeeping genes, RT-PCR, Human primary PBMC.
Beta-2 Microglobulin And Ubiquitin C Identified as Two Robust Housekeeping Genes for RNA Expression Normalization in Real Time PCR on Human Leukocytes and Regulatory T Cells
