No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.

Sepsis is a serious condition caused by a dysregulated immune response of the host triggered by an infection that can potentially lead to malfunction of various organs or even death in severe cases. Some studies have shown that the use of biomarkers could aid in early diagnosis as well as early treatment of sepsis patients. Furthermore, various studies have investigated the idea of using extracellular microRNAs as biomarkers for sepsis diagnosis. This study aimed to see if there were any differences in the quantity and purity of small RNA -which includes microRNA- by performing two different RNA extraction methods (manual and machinery by using a QIAcube) as well as two different volumes by using the ExoRNeasy Serum/Plasma Midi Kit. Blood samples were collected solely from the same self-assessed healthy donor. The plasma samples were frozen and then thawed before the RNA extraction, whether manually or machinery by the QIAcube. The extracted small RNA was then measured for quantity and purity. The quantitative results were analysed by ANOVA followed by post-hoc Tukey test to show the statistically significant difference in the concentration of small RNA. The QIAcube showed higher concentration values compared to the manual method as well as larger initial plasma volume in comparison to the lower initial plasma volume. Meanwhile, the Kruskal-Wallis test showed no statistically significant difference in the purity values among the different methods and volumes. In conclusion, based on this study, the QIAcube could do what human hands do.

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.

Sepsis is a syndrome causing millions of deaths every year. The diagnosis today uses several parameters one of which is blood culture. It can take up to 48 hours to get results. Sepsis can lead to major organ dysfunction in a span of four hours; therefore time is of the essence. Diagnosis of sepsis using multiple biomarkers a “multi marker panel” is currently being studied. One parameter suggested to be included is miRNA. There is need for an efficient extraction method to get the miRNA from the patients’ blood. This can be done by using a kit either manually or in a machine such as the QIAcube. The miRNeasy Serum/Plasma Advanced Kit (Qiagen) was used in both a manual and a QIAcube miRNA extraction with two volumes of plasma (200 μl and 100 μl) from self-assessed healthy individuals. There were 10 extractions made with each method and volume, in total 40 extractions was performed. The miRNA concentration was measure with a Qubit and purity was measured with a nanodrop. The miRNA concentration was statistically significantly higher using the QIAcube 100 μl compared to the other volume and the manual extractions. The turn-around time was shorter with the manual extraction while the hands-on time was shorter in the QIAcube methods. In conclusionthere is still research to be done on both miRNA and miRNA extraction before it can be included in a multi marker panel for sepsis. However the QIAcube does show potential to be used in a clinical laboratory.