A protocol for microRNA extraction from gastrointestinal digesta

Abstract

MicroRNAs (miRNAs) are non-coding RNAs that influence gene-expression via post-transcriptional regulation of target protein-coding RNAs. With literature reports indicating survival of diet-derived miRNAs following their ingestion, it is important to study their stability and concentration during gastrointestinal digestion. The unique combination of chemicals and elevated RNAse content present in the gastrointestinal matrix may be a limiting factor for studying diet-derived miRNAs. First, chemical cross-reactivity with matrix constituents (e.g. bile salts) may interfere with the salt bridge interactions typically present during RNA extraction, reducing the efficiency of the column. Second, high RNAse content may not be fully inhibited during extraction and could continue degrading the miRNAs, as is observed for other tissues with high RNAse content. These combined issues may result in a reduced efficiency in yield and purity of RNA extracts, further limiting the study of diet-derived miRNAs (i.e. in downstream metabolism). In the present manuscript, we display a method based on silica column purification to extract and quantify diet-derived miRNAs from the bioaccessible phase of the gastrointestinal digesta. The proposed protocol provides a simple, quick (less than 2 h), reliable, and systematic method for miRNA purification from gastrointestinal digesta. The optimization showcased that the challenges caused by high RNAse activity, plant bioactive substances and bile-salt content within the gastrointestinal digesta have been overcome and the study of the miRNA fraction in a body fluid so far neglected is now available to researchers, allowing the use of miRNA as biomarkers of intake and potentially biomarkers of biological changes.