Fig. 1

Schematic timeline of all the steps involved in the development of cfRNA biomarkers. (1) Biofluid isolation: after obtaining blood from the patients and centrifugation to get plasma or serum, it is necessary to perform a hemolysis control to measure the contamination by cellular lysis. (2) RNA isolation: prior to the processing of plasma/serum, it is recommended to add external RNA molecules to act as proxies for correct RNA isolation (spike-ins). After isolation of the nucleic acids and before further processing, a step of DNAse digestion is required to limit the contamination of the sample with co-purified DNA. A final step of RNA quantification is required before moving forward to (3) library preparation. To improve reproducibility, a different set of spike-ins are added before starting the process of preparing the RNA for sequencing, and rRNA, lacking biological information, is depleted from the samples. Once the libraries are prepared and quantified, the next step is to sequence them. During this step, it is also possible to add an exogenous library (PhiX), to measure technical variability and ensure reproducibility. (4) Bioinformatic analysis: after the initial quality control of the sample, the data is processed and the expression of several genes linked to a specific phenotype through differential expression analysis or machine learning, to develop a robust biomarker model