Feasibility of miRNA Detection in Particles in Exhaled Air (PExA) using a multiplexed RT-PCR Method

<bold>Background:</bold> MicroRNAs (miRNAs) regulate gene expression in lung physiology and diseases. Frequently protected inside functional extracellular vesicles, they influence gene expression locally and distantly. The increased need to investigate biomolecules' roles in many lung diseases calls for non-invasive approaches, particularly in early identification. miRNA analysis using non-invasive sampling is underreported but promising. <bold>Aim:</bold> Explore the feasibility of analysing microRNAs through the non-invasive method PExA and optimise its application in studying lung diseases. <bold>Methods:</bold> Exhaled particles (PEx) were collected on different membranes to test RNAse activity through a fluorometric test. PEx from four subjects were collected and processed for RNA extraction. miRNA-seq and a multiplex PCR approach (miRCURY LNA miRNA PCR assay, QIAGEN) were used to assess miRNA levels. The PCR assay was designed using a set of genes selected from our previous study. <bold>Results:</bold> We observed elevated RNAse activity in PExA samples, yet identified a membrane where, post-collection, RNAse activity resembles the negative control. miRNA-125b-5p and other miRNAs were reliably detected with both techniques. Synthetic miRNA internal controls in PCR assays ensured consistency. <bold>Conclusions:</bold> We identified a collecting membrane that can be used to reduce the RNAse activity present in PExA samples during collection. A set of miRNAs was detected in these samples using an easy-to-assay RT-PCR-based method, confirming the feasibility of our previously presented detection by RNAseq. These steps mark a significant progression in our ongoing exploration of miRNA roles in lung diseases through the application of PExA.