Abstract

NGS-based Genome-wide Analysis of Short and Long RNAs in Liquid Biopsies: Best Practices for Biomarker Discovery and Validation

Matthias Hackl, CEO and Co Founder, TAmiRNA GmbH

Cell-free RNAs (cfRNAs) encompass small RNAs, such as microRNAs, and long RNA fragments, including mRNAs and long non-coding RNAs. These RNAs are released from cells into biological fluids either actively or passively, where they are stabilized by encapsulation in extracellular vesicles or association with various proteins. Due to their stability and presence in bodily fluids, cfRNAs hold significant potential as disease biomarkers. However, analyzing cfRNAs is challenging due to pre-analytical and analytical variability, which can obscure biological signals. To address this challenge, we have developed and validated an NGS-based workflow for the absolute quantification of microRNAs in liquid biopsies. This method incorporates defined attomolar amounts of synthetic oligonucleotides, known as “spike-ins,” which are designed to be applicable across a diverse range of species and minimize sequencing bias. Our workflow, miND®, has been employed to identify novel organ injury biomarkers, enhancing drug safety monitoring in pre-clinical and clinical trials. Additionally, we have utilized this workflow to study the microRNA cargo in extracellular vesicles produced by mesenchymal stem cells, providing insights into the therapeutically active cargo and the consistency of the molecular composition of EVs across manufacturing batches. Furthermore, we have implemented and validated a workflow for analyzing mRNA and long non-coding RNA fragments in liquid biopsies and extracellular vesicles. Our research has revealed a high complexity of extracellular mRNA fragments, with specific exons (“hot spots”) being particularly enriched. This information has been instrumental in developing targeted assays, such as RT-qPCR, to quantify extracellular mRNAs with high sensitivity and specificity.