Future diagnostic of Sepsis : Biomarker miRSeps-5 manual vs robotic extraction and optimization of miRNA detection by Two-Tailed RT-qPCR technology

Abstract: Sepsis is a syndrome developed in response to infection when a body’s immune system cannot adequately respond to infection. To this day, sepsis holds a peak position in mortality rates worldwide. Currently, SOFA score and blood culturing as means to diagnose sepsis are standard. However, blood culturing is lengthy and sepsis can develop at a faster rate. Faster diagnostic of sepsis in form of biomarker identification directly from the blood is one possible solution. One of the possible biomarker candidate is miRNA, a small non-coding RNA that participates in the regulation of many biological mechanisms of the human body. The aim of this work was to improve extraction of small RNA including miRNA from patient blood plasma via manual extraction using miRNeasy Advanced Kit (Qiagen) and robotic extraction using the same kit and QIAcube machine while comparing two methodologies. All samples containing extracted small RNA including miRNA were analysed with a Qubit 4.0 (Thermo Fisher Scientific) and DS-11+spectrophotometer (DeNovix). Furthermore, two-tailed RT-qPCR was investigated for potential to quantify synthetic and naturally occurring miRSeps-5 extracted from blood plasma. PCR System Machine was used to obtain Cq-values and melting curves. In order to use absolute quantification to quantify microRNA standard curve was optimized achieving a 116% efficiency and correlation coefficient R2=0.97. This work shows encouraging results of using two-tailed RT-qPCR to detect miRSeps-5 extracted from human plasma for future biomarker research.