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Background  Technical feasibility of RNA quantification by real time RT-PCR has led to enormous utilization of this method. However, real time PCR results need to be normalized due to the high sensitivity of the method and also to eliminate technical variation. Normalization against a reference gene that is constitutively transcribed and has minimum variation among samples is the ideal method. Nevertheless, many studies have shown that there is no general reference gene(s) with ideal characteristics and candidate reference genes should be tested before being used as a “normalizer” in each study.    Methods  The current study investigated the effects of previous exposure of the host to experimental test antigens and culturing time on the expression of 11 candidate genes when blood mononuclear cells (BMCs) were cultured and treated  in-vitro  by hen egg white lysozyme,  Candida albicans  extract and a mitogen. Mononuclear cells were isolated and cultured from 12 bovine blood samples representing 3 different immunological statuses. The expression of candidate housekeeping genes were measured by real-time RT-PCR at 4 and 24 hours post culture. The expression of candidate genes were first compared between the two time points in untreated samples. Constitutively expressed genes were further tested in linear mixed effects models to examine the effect of previous host exposure and  in-vitro  treatments.    Results  Our findings showed that the expression of the most common reference genes,  β-actin,  and  Glyceraldehydes-3-phosphate dehydrogenase  (GAPDH), are significantly decreased at 24 hours after culturing BMCs, even without any treatment. The effect of culturing time was also significantly influenced the expression of  18s ribosomal RNA ,  β2-microglobulin ,  Tyrosine 3-monooxygenase/tryptophan 5-monoxygenase activation protein, zeta polypeptide  (YWHAZ) in BMCs. Only the expression of  C-terminal binding protein 1  (CTBP1) and  RAD50  among all tested genes were consistent after treatment of cultured BMCs with  C. albicans  whole yeast extract and Hen Egg White Lysozyme (HEWL), respectively. In addition, expressions of CTBP1, and RAD50 were independent from previous exposure of the host to the antigen.    Conclusions  The results of this study demonstrated inconsistent expression of commonly used reference genes in untreated cultured BMCs over time. As this condition applies to negative controls in real time RT-PCR study designs, normalization against these genes can largely deceive the outcome, especially in kinetic studies. Moreover, the potential effects of immunological memory on the expression of reference genes should be considered if BMCs are collected from different individuals under different environmental conditions and if these cells are treated  in-vitro  by an antigen.

bmc immunology

The effect of immunological status, in-vitro treatment and culture time on expression of eleven candidate reference genes in bovine blood mononuclear cells