RNA normalization

With the increased interest in gene marker based diagnosis, the choice of housekeeping genes for the various assays becomes an important consideration that could affect sensitivity, specificity, and across laboratory repro-ducibility of results. In our Laboratory, we performed a detailed examination of 11 available housekeeping genes in peripheral blood of healthy volunteers before selecting a suitable housekeeping gene for our assay (Sabek et al., 2002). Expression of the following housekeeping genes was evaluated: 18S rRNA (18S), acidic ribosomal protein (PO), beta-actin (PA), cyclophilin (CYC), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerokinase (PGK), p2-microglobulin (p2m), p-glucuronidase (GUS), hypoxanthine phosphoribosyl transferase (HPRT), transcription factor IID, TATA-binding protein (TBP), transferring receptor (TfR). The results of indicated that 18S, P-actin, and P-glucuronidase have the least variability of expression over time and are more reliable for normalizing our data. Surprisingly, other housekeeping genes used in previous human studies, such as GAPDH and cyclophilin, had higher variability between specimens and generated irreproducible results. The lack of reliability of GAPDH as a housekeeping gene has been observed by others as well (Tricarico et al., 2002; Bustin, 2000). Hoffmann et al. (2003) has shown that 18s RNA was an appropriate candidate housekeeping control for transcript normalization since its expression levels were not found to vary among different tissues, cell types, with or without stimulation, or from experimental treatments. In contrast GAPDH and p-actin expression varied widely, exhibiting Ct fluctuations and nearly 10-fold differences in some activated cell populations. In addition, cyclophilin is not a practical calibrator for transplantation-related studies due to its direct role in the calcineurin pathway affected by standard immunosuppressive therapies. GUS was found to be a fairly constant and reliable expression target. However, the level of GUS transcript expression may compete with several immune targets and, therefore, does not allow for multiplex assays and could invalidate quantitative precision. Based on these findings 18s

RNA was regarded as the most reliable internal housekeeping control for analyses. However, it is vital to test for the effect of different drugs used on the expression levels of the housekeeping genes if we are to incorporate this technique as standard clinical tool for monitoring allograft dysfunction and guiding individualized immunotherapeutic interventions. Further discussion of the choice of a normalizer gene is presented in Chapter 4.

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