Extraction of miR-223 from human blood plasma and quantification using the two-tailed RT-qPCR and absolute quantification

Sammanfattning: Sepsis is a very dangerous and life-threatening disease that develops when the body’s reaction to infection causes damage to the body’s tissues and organs. It is difficult to diagnose it and it develops fast leading to a high mortality rate. Current methods rely on blood culturing and multiple biomarkers, such as C-reactive protein and procalcitonin, that take too long to produce results. A possible solution to this problem lies in specific biomarkers such as microRNAs, which are small non-coding single stranded RNA molecules that contain around 22 nucleotides and have a big role in regulating gene expression. Being specific biomarkers for particular disease makes microRNAs promising biomarkers for sepsis. The aim of the project was to optimize the process from extraction to quantification of microRNAs using the miRNeasy Serum/Plasma Advanced Kit-Qiagen Kit (manual) and to see if the Two-tailed RT-qPCR (TATAA Biocenter) technique could quantify the samples. Blood plasma from healthy donors was used for microRNA extractions and was separated into two categories- spiked-in samples and non-spiked samples. Spiked-in samples were spiked with a synthetic microRNA- miR-223 and served as a positive control. All samples were quantified using the absolute quantification and the Two-tailed RT-qPCR method (TATAA Biocenter). Quantification was successful for all samples showing that the method was optimized, parameters for optimization were within the wanted range, and quantifiable. More research is needed, however, the method has potential in becoming a simple and quick novel tool in diagnosing sepsis in the early stages and thus saving lives.