Assessment of methods for microRNA isolation, microRNA amplification, and development of a normalization strategy for sepsis biomarker research

Abstract: Sepsis, defined by organ dysfunction caused by an adverse immune response of the host to an infection, comes with considerable cost in human lives and as a substantial burden financially. Significant upgrades have been made over the past two decades when diagnosing and treating sepsis but still with room for improvements. Early detection is a cornerstone in the fight against sepsis, and the focus on strengthening diagnostics is in the forefront of modern research. The implementation of biomarkers may be the path of progression in this objective. This study aimed at establishing procedural foundations when using microRNAs as potential biomarkers. The study conducted looked at: (1) Isolation procedure, of microRNA from human plasma, of three kits: Total RNA Purification Kit (Norgen Biotech), miRNAeasy Serum/Plasma Kit (Qiagen), and miRNeasy Serum/Plasma Advanced Kit (Qiagen). (2) Amplification of miRNA through two Reverse Transcription Quantitative PCR methods: Two-tailed RT-qPCR (TATAA Biocenter), and miRCURY LNA miRNA PCR (Qiagen). (3) Developing a normalization strategy by identifying miRNA reference targets in a geNorm pilot experiment. Qubit analysis revealed that the two isolation kits from Qiagen performed similar, and better that the Norgen kit. The Two-tailed RT-qPCR failed to amplify miRNA samples, whereas the miRCURY LNA miRNA PCR showed consistent amplification across samples with a high call rate. The geNorm analysis concluded that hsa-miR-425-5p and hsa-miR-93-5p was the optimal reference target set. The study demonstrated that the isolation kits from Qiagen coupled with the miRCURY LNA miRNA PCR is a viable option for future miRNA biomarker studies.