Computational protocol: Potential blood based markers of celiac disease

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Protocol publication

[…] Potential reference genes for the mRNA analysis were investigated using a Human Endogenous Control Plate (Life Technologies) containing assays for 32 potential reference genes, and cDNA from a total of nine blood RNA samples including three samples from cases with no mucosal injury (Marsh 0) and six with varying degrees of mucosal injury (Marsh 2-3C). Three potential reference genes (Additional file ) were selected based on low sample-to-sample variation in mRNA levels, as investigated using the NormFinder algorithm [] in version 5.4.2 of the Genex software package (MultiD Analyses, Göteborg, Sweden). The three selected reference genes were analyzed in the complete dataset, and the final selection of reference gene/s was established using the complete dataset by means of both low sample-to-sample variation and an absence of group differences in expression.Genes for analysis of mRNA and protein levels were selected by reviewing published studies on blood mRNA/protein expression in CD, and by functional context (Additional file ). [...] Version 10 of the STATISTICA software package (StatSoft, Tulsa, OK) was used in all statistical analyses. Differential expression was investigated using Kruskal–Wallis one-way analysis of variance by ranks, except for the analysis of differential expression in cases stratified based on having one or two DQB1*02 alleles, where the Mann–Whitney U test was used. Post-hoc comparisons of mean ranks of all pairs of groups were performed (significance level p < 0.05, two-sided significance levels with a Bonferroni adjustment) []. Spearman rank correlation was used to investigate relations between mRNA/protein levels and histopathology and CD risk gradient based on HLA type. For all statistical analyses, except the post-hoc comparisons, a false-discovery rate was used and set to 5% [].The diagnostic performance of individual assays and logistic regression models of assay combinations was evaluated using receiver operating characteristic (ROC) curve analysis (MedCalc Statistical Software version 13.1.2, MedCalc Software, Ostend, Belgium). […]

Pipeline specifications

Software tools NormFinder, Statistica, MedCalc
Applications Miscellaneous, qPCR
Diseases Celiac Disease