Variability of cytokine gene expression in intestinal tissue and the impact of normalization with the use of reference genes.
Variability of cytokine gene expression in intestinal tissue and the impact of normalization with the use of reference genes.
复制标题
肠道组织中细胞因子基因表达的变异性以及使用参考基因标准化的影响。
DOI：
10.1016/j.cyto.2014.08.010
复制
发表时间：
2015
期刊：
影响因子：
3.8
通讯作者：
Richardson-Harman,Nicola
中科院分区：
文献类型：
--
作者：
McGowan,Ian;Janocko,Laura;Burneisen,Shaun;Bhat,Anand;Richardson-Harman,Nicola
ObjectiveTo determine the intra- and inter-subject variability of mucosal cytokine gene expression in rectal biopsies from healthy volunteers and to screen cytokine and chemokine mRNA as potential biomarkers of mucosal inflammation.Design and methodsRectal biopsies were collected from 8 participants (3 biopsies per participant) and 1 additional participant (10 biopsies). Quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to quantify IL-1β, IL-6, IL-12p40, IL-8, IFN-γ, MIP-1α, MIP-1β, RANTES, and TNF-α gene expression in the rectal tissue. The intra-assay, inter-biopsy and inter-subject variance was measured in the eight participants. Bootstrap re-sampling of the biopsy measurements was performed to determine the accuracy of gene expression data obtained for 10 biopsies obtained from one participant. Cytokines were both non-normalized and normalized using four reference genes (GAPDH, β-actin, β2 microglobulin, and CD45).ResultsCytokine measurement accuracy was increased with the number of biopsy samples, per person; four biopsies were typically needed to produce a mean result within a 95% confidence interval of the subject’s cytokine level approximately 80% of the time. Intra-assay precision (% geometric standard deviation) ranged between 8.2 and 96.9 with high variance between patients and even between different biopsies from the same patient. Variability was not greatly reduced with the use of reference genes to normalize data.ConclusionsThe 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.
