Evaluation of the Two-tailed RT-qPCR method with the application of manual vs robotic extraction of miRNA

Sepsis is aggressive and severe inflammatory body response to an infection and is considered to be one of the most common death causes in patients. The current diagnosis of sepsis is not fast enough to help those who get sepsis, due to its fast progression. The current golden standard for sepsis diagnosis is blood culturing. However, the biggest downside of it is the long time. Research is now focused on finding a faster way to diagnose sepsis on early stages. The most promising one tends to be the usage of biomarkers. Today, there are 260 defined sepsis biomarkers, however, only few of them are clinically used. Among them, C-reactive protein, and procalcitonin. Another potential biomarker could be miRNAs. The research about that today is at early stage. To use miRNAs as biomarkers, they need to be quantified. One way to quantify miRNAs is the two-tailed RT-qPCR method together with absolute quantification. This study focused on evaluating the best extraction method of small RNA for later quantification of specific miRNA. The blood plasma from healthy donors was divided into spiked and non-spiked samples, where the synthetic miRSeps-3 served as a spike-in positive controls. All samples were extracted using two methods, manual and robotic with Qiacube (Qiagen). Absolute quantification was applied to quantify miRNA in all samples. The successful results indicated that the two-tailed RT-qPCR was sensitive enough. More optimization is required for the methods, however, the whole method has a good potential to become helpful for clinical usage in the futur