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

Johannes Grillari,Amy Schofield ,Regina Grillari,Christopher E. Goldring,Susanna Skalicky,Nina Buchtele,Christian Schoergenhofer,Teresa L. Krammer,Klemens Ruprecht,Bernhard J. H. Frank,Aaron T. Smith ,Moritz Weigl,Warren E. Glaab,Matthias Hackl,Kseniya Khamina,Marianne Pultar,Carolin Otto,Andreas Diendorfer ,Jochen G. Hofstaetter,Hubert Rehrauer,Catharine Aquino Fournier,Bernd Jilma

doi:10.3390/ijms23031226

Abstract

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 microRNA Next-Generation-Sequencing Discovery 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.

Highlights

MicroRNAs are small endogenous non-coding RNAs of 17–25 nucleotides in length that regulate gene expression in mammalian cells
Three of the selected kits, CleanTag, NEXTFLEX and QIAseq, relied on sequential 50 and 30 adapter ligation to microRNAs followed by reverse transcription and PCR amplification
NEXTFLEX and QIAseq, used random nucleotide sequences that serve as unique molecular indices (UMIs)

Summary

Introduction

MicroRNAs (miRNAs) are small endogenous non-coding RNAs of 17–25 nucleotides in length that regulate gene expression in mammalian cells. MicroRNAs are produced by virtually all cell types, and their expression can be changed in response to physiological stimuli and pathological processes. Multiple studies indicated that levels of microRNAs can be used as diagnostic and prognostic biomarkers for various diseases including cardiovascular disease [4], infectious disease [5] and cancer [6,7]. MicroRNAs have drawn much attention as promising diagnostic, disease severity and prognostic biomarkers [8,9]. The minimally invasive analysis of microRNAs in liquid biopsies allows for rapid, economical, and repeated sampling. This provides an opportunity for the development of screening programs and close monitoring of treatment response and disease progression [9]

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