Current challenges and best practices for cell-free long RNA biomarker discovery

Abstract

The analysis of biomarkers in biological fluids, also known as liquid biopsies, is seen with great potential to diagnose complex diseases such as cancer with a high sensitivity and minimal invasiveness. Although it can target any biomolecule, most liquid biopsy studies have focused on circulating nucleic acids. Historically, studies have aimed at the detection of specific mutations on cell-free DNA (cfDNA), but recently, the study of cell-free RNA (cfRNA) has gained traction. Since 2020, a handful of cfDNA tests have been approved for therapy selection by the FDA, however, no cfRNA tests are approved to date. One of the main drawbacks in the field of RNA-based liquid biopsies is the low reproducibility of the results, often caused by technical and biological variability, a lack of standardized protocols and insufficient cohorts. In this review, we will identify the main challenges and biases introduced during the different stages of biomarker discovery in liquid biopsies with cfRNA and propose solutions to minimize them.

Integrated analysis of circulating cell free nucleic acids for cancer genotyping and immune phenotyping of tumor microenvironment

“…Furthermore, the input quality also serves as a critical parameter for the quality assessment of the data. RNA sequencing is an extensively used tool for estimating gene expression, however it does not provide absolute quantification of the data like RT-qPCR methods ( Cabús et al, 2022 ).…”

Enhancing clinical potential of liquid biopsy through a multi-omic approach: A systematic review

“…RNA-seq of single prostate CTCs from patients resistant to Androgen Receptor (AR) inhibitor showed activation of noncanonical Wnt signaling and low glucocorticoid receptor expression compared with untreated cases (Miyamoto et al, 2015). Furthermore, the transcriptomic profile of CTCs was analyzed to identify a specific subpopulation involved in the metastatic spread and organotropism of different cancer types (Giuliano et al, 2014;Castro-Giner and Aceto, 2020;Schuster et al, 2021;Yu et al, 2021).…”

“…This can be partly explained by the differences in the samples tested, in terms of ethnicity of the courts, the use of endogenous controls, the biofluid analyzed, but above all on the detection method used. Consequently, standardization of procedures is needed to take full advantage of miRNAs as cancer biomarkers ( Cabús et al, 2022 ).…”

Depletion-assisted multiplexed cell-free RNA sequencing reveals distinct human and microbial signatures in plasma versus extracellular vesicles

“…However, miRNAs represent only a small proportion of the human transcriptome [10]. Therefore, a broader space of cfRNAs, such as messenger RNAs (mRNAs), long non-coding RNAs (lncRNAs), and circular RNAs (circRNAs) have started to be investigated later [4][5][6][7]11]. Due to RNases in human blood, these cell-free long RNA species (>50 nt) have relatively low concentrations.…”