Supplementary Material for: CRITICAL STEPS FOR HUMAN GUT EXFOLIOME RNA PROFILING ANALYSIS USING NON-INVASIVE STOOL SAMPLES

Abstract Introduction: Dietary exposure and drug treatments influence gut cellular pathways and hence growth and potentially even the gut-brain-microbiome axis. Since eukaryotic mRNA presents poly A sequence that distinguishes them from the prokaryotes mRNA, we could analyze the gene expression of human gut cells using exfoliated gut cells available in stool samples. However, the impact of the critical steps of these non-invasive methods must be analyzed. Methods: We tested prokaryote contamination in all the steps of different procedures to analyze human exfoliome by microarrays and the influence of the fecal sampling collection process. Results & Conclusion: The least bacterial contamination was found using RNA amplified with oligo dT from GeneChip 3´ IVT Pico Reagent kit or using RNA purified by both Oligotex® + oligodT. RNA later® collection of feces affects the microarray results compared to directly frozen fecal samples, although both methods produce similar cDNA quality. This technique is a potential non-invasive diagnostic tool that can be applied to larger studies to quantify intestinal gene expression in humans with non-invasive samples, but samples should always be collected and analyzed under the same procedure.