An Optimized and Improved Protocol for Efficient Isolation of Extracellular RNA from Human Serum

The extraction of extracellular RNA from human serum is challenging due to its low abundance, high degree of fragmentation, and susceptibility to rapid degradation. These limitations are further compounded in pediatric research, where low blood sample volume is a major, frequent constraint, necessitating highly sensitive and efficient RNA extraction methods suitable for minimal-input samples. Existing protocols use TRIzol or commercial kits, which are primarily optimized for the enrichment of small RNAs rather than for the comprehensive recovery of extracellular RNA. Here, we present an optimized protocol for efficient isolation of extracellular RNA from human serum, using a modified TRIzol-based method following chloroform phase separation. We processed serum samples from pediatric patients using TRI-Reagent-based lysis and chloroform phase separation, and then compared the results of subsequent isopropanol precipitation (protocol 1) and silica-column-based purification (protocol 2). Protocol 2 significantly improved RNA yield (5-fold), purity (A₂₆₀/A₂₈₀ and A₂₆₀/A₂₃₀ ratios), and integrity compared to protocol 1, as confirmed by agarose gel electrophoresis. The extracted RNA was successfully utilized for cDNA synthesis and qRT-PCR amplification of the human CRBN gene, demonstrating its suitability for downstream applications. Protocol 2 provides a rapid, cost-effective, and robust approach for isolating extracellular RNA from serum, validated primarily for RT-PCR-based applications, yielding RNA of improved quantity and purity suitable for biomarker analysis and gene expression studies from minimal-input clinical samples. © 2026 Wiley Periodicals LLC. Basic Protocol: Optimized isolation of extracellular RNA from human serum using TRIzol-based lysis and chloroform phase separation Support Protocol 1: Isopropanol-precipitation-based recovery of extracellular RNA from aqueous phase Support Protocol 2: Silica-column-based purification of extracellular RNA for enhanced yield and purity Support Protocol 3: cDNA synthesis and qRT-PCR analysis of extracellular RNA.