Optimization of the Two-Tailed RT qPCR method with synthetic miR-16 and miR-210 in human plasma for future diagnostics of sepsis

Sammanfattning: Every year, around 6 million people lose the fight against sepsis. Sepsis is a dysregulated response from the host to an infection, leading to life-threatening organ dysfunction. The gold-standard diagnostics of sepsis to date is blood culturing and the results take up to three days to be validated Therefore, it is important to focus on biomarkers like MicroRNA [miRNA] to develop faster diagnostics tools as they have previously shown to be potential minimally-invasive biomarkers for many diseases of different origin. RT-qPCR is known to amplify DNA, while a new primer, called the two-tailed primer, was developed by TATAA Biocenter to specialize on miRNA amplification. The aim of this study is to perform the two-tailed RT-qPCR method manually with two synthetic miRNAs, miR-210 and miR-16, reduce the amount of plasma to a minimum of 100 μl and optimize this method for the use in conventional labs. Whole blood was drawn, centrifuged and the resulting plasma underwent RNA extraction. Synthetic miRNAs were used for spiking and the two-tailed RT-qPCR method was performed and both melt curve analysis, standard curve analysis and absolute quantification were performed. Amplification was detected in all samples, allowing to conclude that the two-tailed primers work. The amplification efficiencies and the linearity for both synthetic miRNAs were determined at 77% and 0.99, respectively. Absolute quantification showed promising quantification for all spiked samples while it needs to be taken into account that the amplification efficiencies were rather low and the standard curves used for absolute quantification should be diluted with care.