B-235 Utility of microRNA Extracted with the miRNA Enhance Buffer in Next-Generation Sequencing

Abstract Background MicroRNAs (miRNAs) play an important role in the regulatory mechanisms of posttranscriptional gene expression and have garnered attention in clinical laboratories as liquid biopsies. miRNAs are approximately 20–25 nucleotides in length, which is shorter than mRNA, and are less susceptible to nucleases, thereby providing a distinct advantage in specimen preservation and stability. In the diagnostic application of miRNAs, the amount and quality of the extracted nucleic acids are crucial factors that affect the feasibility, accuracy, and reliability of downstream analysis. Ensuring these factors through the selection and optimization of extraction methods is crucial to further increase the clinical utility of miRNAs. We evaluated the effectiveness of the miRNA Enhance Buffer (KANTO CHEMICAL Co., Inc .) using several commercial RNA extraction reagents and miRNA-specific extraction reagents for both cell lines and clinical samples. Moreover, we examined its performance in next-generation sequencing (NGS) analysis. Methods This study used K562 cell line pellets prepared from 10^6 cells . miRNA extraction was performed using the following four protocols: (A) the Cica Geneus RNA Prep Kit (for Tissue) 2 (KANTO CHEMICAL CO., INC.) and the miRNA Enhance Buffer; (B) the High Pure miRNA Isolation Kit (Roche); (C) the miRNeasy Mini Kit (QIAGEN) and the miRNA Enhance Buffer; and (D) the miRNeasy Mini Kit. In protocols (A) and (C), 70% and 100% ethanol were replaced with the miRNA Enhance Buffer to bind nucleic acids to the columns, and all subsequent steps were performed according to the manufacturer’s instructions. In protocols (B) and (D), miRNA extraction was performed according to the manufacturer’s instructions. Libraries were prepared using the QIAseq miRNA UDI Library Kit (QIAGEN), and NGS analysis was performed using the NovaSeq X Plus system (Illumina). For data analysis, the RNA-seq Analysis Portal (QIAGEN) was used. Results All extraction methods recorded high Q scores above 40, with the read counts being highest for protocol (A), and annotations to the miRBase were also highest for protocol (A). Spearman’s rank correlation analysis revealed a strong correlation between all the protocol s, and the volcano plot showed some miRNAs, but no consistent trend was observed. Conclusion In conclusion, nucleic acids extracted with the miRNA Enhance Buffer are viable for NGS analysis, and the miRNA Enhance Buffer exhibits similar performance to that of other reagents.