title: Variability of cytokine gene expression in intestinal tissue and the impact of normalization with the use of reference genes.
abstract: OBJECTIVE
  To 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.
  
  
  METHODS
  Rectal 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).
  
  
  RESULTS
  Cytokine 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Magee-Womens Research Institute, Division of Gastroenterology, Hepatology, and Nutrition, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA. Electronic address: imcgowan@pitt.edu.
authors.forename: Ian
authors.initials: I
authors.lastname: McGowan
authors.affiliation: Magee-Womens Research Institute, Division of Gastroenterology, Hepatology, and Nutrition, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Laura
authors.initials: L
authors.lastname: Janocko
authors.affiliation: Magee-Womens Research Institute, Division of Gastroenterology, Hepatology, and Nutrition, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA.
authors.forename: Shaun
authors.initials: S
authors.lastname: Burneisen
authors.affiliation: Case Western Reserve University at MetroHealth Medical Center, Cleveland, OH, USA.
authors.forename: Anand
authors.initials: A
authors.lastname: Bhat
authors.affiliation: Alpha StatConsult, LLC, Damascus, MD, USA.
authors.forename: Nicola
authors.initials: N
authors.lastname: Richardson-Harman
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D001706:Biopsy; D018925:Chemokines / Q000235:genetics*; D016207:Cytokines / Q000235:genetics*; D000068598:Data Accuracy; D015870:Gene Expression; D020043:Genes, Essential; D064368:Healthy Volunteers; D006801:Humans; D007413:Intestinal Mucosa / Q000276:immunology* / Q000378:metabolism; D008875:Middle Aged; D012333:RNA, Messenger / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D012007:Rectum / Q000276:immunology* / Q000378:metabolism; D012015:Reference Standards; D015203:Reproducibility of Results
chemical_list: D018925:Chemokines; D016207:Cytokines; D012333:RNA, Messenger
keywords: Gut-associated lymphoid tissue (GALT); Housekeeping genes; Intestinal cytokine gene expression; Reference genes