Högskolan i Skövde

Sepsis presents a complex challenge, requiring detailed investigation into its immunopathology, diagnosis and treatment. Among 258 promising biomarkers for sepsis diagnosis, miRNAs are emerging as valuable components to be incorporated in a multimarker panel for sepsis assessment when assessing bodily fluid samples. The aim of this project was to compare manual and semi-robotic techniques for extracting total RNA, including miRNA, from healthy human blood plasma samples. The comparison focused on hands-on time (HOT), turn-around time (TAT), and the absolute quantification of the candidate miRNA, miR-Seps5. The study utilized two extraction methods to isolate total RNA, including miRNA. The emphasis was on detecting and quantifying the novel candidate miRNA, miR-Seps5, using the Two-Tailed RT-qPCR technique and absolute quantification. This approach effectively amplified synthetic and circulating miR-Seps5 from 100 μl of healthy human blood plasma. No-significant difference was found between two methods of extraction on the basis of TAT and data retrieved from two-tailed RT-qPCR while HOT was significantly lower for semi-robotic extraction method as compared to manual extraction method indicating Qiacube to be a time efficient method. Linear standard curve and melt curve analysis provided insights into the specificity of Two-tailed RT-qPCR assay but unspecified amplification in non-template control (NTC) requires further investigation. The quantified values of miR-Seps5 in both spiked and non-spiked samples showed inconsistency. Considering these inconsistencies, further analysis is required to assess the reliability of the RT-qPCR assay and miR-Seps5 before considering it as a suitable biomarker in a future multimarker panel for sepsis diagnosis.