Commonly used reference genes are actively regulated in in vitro stimulated lymphocytes

Quantitative polymerase chain reaction (Q-PCR) studies of urine sediments have demonstrated an increased expression of cytotoxin genes during episodes of acute rejection of renal allografts. To compensate for differences in initial sample size and cDNA preparation, standard Q-PCR experiments involve normalization to a reference gene. Although stable expression of reference genes is a prerequisite for any Q-PCR analysis, commonly used reference genes have demonstrated a varying expression across tissues and various stimuli. In this study, cellular expression of several reference genes was investigated in a mixed lymphocyte reaction as a model of gene expression during alloreactive T-lymphocyte activation and acute rejection. Gene expression was quantified using Q-PCR, normalized to cell counts obtained by DNA quantification and corrected for cell polyploidy using flow cytometry. Examined reference genes were 18S rRNA, beta-actin (ACTB), hydroxymethylbilane synthase (HMBS), hypoxanthine phosphoribosyltransferase (HPRT1) and peptidylprolyle isomerase B (PPIB). This study also examined two novel T-lymphocyte-specific reference genes: CD3E and CD8B. HMBS and HPRT were 18.8- and 7.4-fold upregulated, respectively, ACTB was 5.3-fold upregulated, PPIB was 3.2-fold upregulated while 18S rRNA remained stably expressed. The T-lymphocyte-specific reference gene CD3E remained stable while CD8B was upregulated 2.3-fold. In conclusion, several commonly used reference genes were actively regulated during alloreactive T-lymphocyte activation. Additionally, we introduce two stable T-lymphocyte-specific reference genes that might be useful in a Q-PCR analysis of T-lymphocyte-specific cytotoxin genes in urine sediments, as they overcome the contribution of reference gene mRNA from cells irrelevant for diagnosis.