Variability of cytokine gene expression in intestinal tissue and the impact of normalization with the use of reference genes

The number of biopsy samples required to provide an accurate result varied by target although 4 biopsy samples per subject and timepoint, provided for >77% accuracy across all targets tested. Biopsies within the same subjects and between subjects had similar levels of variance while variance within a biopsy (intra-assay) was generally lower. Normalization of inflammatory cytokines against reference genes failed to consistently reduce variance. The accuracy and reliability of mRNA expression of inflammatory cytokines will set a ceiling on the ability of these measures to predict mucosal inflammation. Techniques to reduce variability should be developed within a larger cohort of individuals before normative reference values can be validated.