Evaluation of Plasma-Derived Cell-Free RNA Isolation Methods Using PCR- Based Quantification

Background: RNA biomarker research has grown significantly in recent years, with a focus on blood-based biomarker studies using cell-free nucleic acids (cf-NAs). These RNA derivatives are useful in diagnostic procedures for genetic changes and can be found in a variety of physiological fluids, such as plasma, brain fluid, and urine. RNA biomarkers are composed of different coding and non-coding transcripts. Among these, cfRNAs extracted from blood provide a minimally invasive source for disease diagnosis and monitoring. However, cfRNA isolation is challenging due to degradation, instability, lack of standardization, and contamination by microbial, environmental, and intrasample DNA. Therefore, it is crucial to obtain cell-free RNAs in high concentration using appropriate methods and process them using downstream PCR systems. ​​Methods and Results: To compare the purity and quality of cfRNAs isolated from plasma, five different isolation methods using commercial cfRNA kits were evaluated by digital PCR (dPCR) and qRT-PCR. RNA quality was assessed across kits, and expression levels were measured using cfRNA reference genes such as GAPDH and B2M. The results of analysis revealed that the GAPDH housekeeping gene showed greater consistency than B2M, and both genes exhibited statistically significant expression. Conclusions: Our comparative analysis of five cfRNA isolation methods demonstrated that commercial kits outperformed phenol–chloroform–based procedures. These results underscore the need for optimized isolation strategies and careful reference gene selection to enhance the reliability of cfRNA-based biomarker discovery.