The Role of Cell-Free RNA in Clinical Diagnosis and Evaluation of Cell-Free RNA Extraction Methods

Cell-free RNA (cf-RNA) is released from cells and circulated in the body via the circulatory and endocrine systems. Subsequently, cf-RNA can be extracted from different biofluids by various extraction methods. This review aims to: (1) clarify the clinical role of cf-RNA and methods of extraction, (2) introduce the evaluation parameters and methods, (3) summarize the evaluation of several common extraction methods and propose optimizing solutions for them. Eighty articles on the application of cf-RNA in diagnosing diseases and cf-RNA extraction published in journals with good impact factor (higher than 2) were then summarized and sorted into groups based on their targeted disease: (1) Cancers and (2) Neurodegenerative diseases since the cf-RNA is being studied mainly in these two fields. The general design of these studies is comparing cf-RNA to the current means of diagnosis, which are considered as standards such as endoscopy, biopsy, CSF analysis. By quantifying cf-RNA using qRT-PCR, previous studies were able to detect several cancers and neurodegenerative diseases. The key advantage of using the cf-RNA as a biomarker is reducing the risks and invasiveness without losing sensitivity since it can be extracted in the peripheral area. Furthermore, cf-RNA expression levels vary according to disease stages, suggesting its potential as prognostic markers. The main challenge in using cf-RNA is achieving the optimal yield, purity, integrity with minimal contamination. The current methods like Trizol LS, miR-Neasy, mirVANA have yielded unsatisfying results; therefore, they require modifications to optimize their results. miRCURY kit has the highest RNA yield but has been discontinued. In conclusion, cf-RNA holds excellent promises as a promising biomarker in the future. However, due to certain challenges, the use of cf-RNA is currently limited to clinical studies due to the instability in yield and integrity, requiring supporting studies and extraction methods evaluation.