A MicroRNA Next-Generation-Sequencing Discovery Assay (miND) for Genome-Scale Analysis and Absolute Quantitation of Circulating MicroRNA Biomarkers

Khamina, Kseniya, Diendorfer, Andreas B, Skalicky, Susanna, Weigl, Moritz, Pultar, Marianne, Krammer, Teresa L, Fournier, Catharine Aquino, Schofield, Amy L, Otto, Carolin, Smith, Aaron Thomas
et al (show 12 more authors) (2022) A MicroRNA Next-Generation-Sequencing Discovery Assay (miND) for Genome-Scale Analysis and Absolute Quantitation of Circulating MicroRNA Biomarkers. INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES, 23 (3). 1226-.

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The plasma levels of tissue-specific microRNAs can be used as diagnostic, disease severity and prognostic biomarkers for chronic and acute diseases and drug-induced injury. Thereby, the combination of diverse microRNAs into biomarker signatures using multivariate statistics seems especially powerful from the perspective of tissue and condition specific microRNA shedding into the plasma. Although next-generation sequencing (NGS) technology enables one to analyse circulating microRNAs on a genome-scale level, it suffers from potential biases (e.g., adapter ligation bias) and lacks absolute transcript quantitation as well as tailor-made quality controls. In order to develop a robust NGS discovery assay for genome-scale quantitation of circulating microRNAs, we first evaluated the sensitivity, repeatability and ligation bias of four commercially available small RNA library preparation protocols. The protocol from RealSeq Biosciences was selected based on its performance and usability and coupled with a novel panel of exogenous small RNA spike-in controls to enable quality control and absolute quantitation, thus ensuring comparability of data across independent NGS experiments. The established <b>mi</b>croRNA <b>N</b>ext-Generation-Sequencing <b>D</b>iscovery Assay (miND) was validated for its relative accuracy, precision, analytical measurement range and sequencing bias and was considered fit-for-purpose for microRNA biomarker discovery. Summarized, all these criteria were met, and thus, our analytical platform is considered fit-for-purpose for microRNA biomarker discovery from biofluids in the setting of any diagnostic, prognostic or patient stratification need. The established miND assay was tested on serum, cerebrospinal fluid (CSF), synovial fluid (SF) and extracellular vesicles (EV) extracted from cell culture medium of primary cells and proved its potential to be used across different sample types.

Item Type: Article
Uncontrolled Keywords: biomarkers, drug safety, microRNA, next-generation sequencing, small RNA-sequencing, spike-in, toxicology
Divisions: Faculty of Health and Life Sciences
Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 26 May 2022 09:06
Last Modified: 03 Feb 2024 08:28
DOI: 10.3390/ijms23031226
Open Access URL: https://doi.org/10.3390/ijms23031226
Related URLs:
URI: https://livrepository.liverpool.ac.uk/id/eprint/3155519