Sepsis is a life-threatening condition in which a dysregulated immune response causes damage to the patient’s organs. The condition moves fast leaving little time for diagnosis and starting treatment. Early diagnosis would help save lives and reduce the amount of antibiotics used. The aim of this study was to compare manual and robotic extractions for miRNA extraction and to quantify miR-seps4, a possible biomarker for sepsis, from human plasma. During the first part of the study manual extractions done by hand and robotic extractions done using QIAcube (Qiagen) of miRNA using the miRNeasy Serum/Plasma Advanced Kit (Qiagen) were compared using the quantity and purity of RNA they produced. Then two-tailed RT-qPCR was optimised for human plasma spiked with synthetic miR-seps4. The final objective was to use these methods to test whether two-tailed RT-qPCR could be used to identify and quantify miR-seps4 from non-spiked human plasma using the absolute quantification method. The QIAcube (Qiagen) extractions gave a higher concentration of RNA while manual RNA extractions had a higher purity A successfully optimised standard curve was made using synthetic miR-seps4 though the melt curve showed contamination and possible non-specific products. Non-spiked RNA extractions showed successful amplification of miR-seps4 that could be quantified through the absolute quantification method. This makes miR-seps4 a good target for further research and a possible addition to a multi-marker panel for early diagnosis of sepsis.
| Identifer | oai:union.ndltd.org:UPSALLA1/oai:DiVA.org:his-21656 |
| Date | January 2022 |
| Creators | Asikainen, Anniina |
| Publisher | Högskolan i Skövde, Institutionen för biovetenskap |
| Source Sets | DiVA Archive at Upsalla University |
| Language | English |
| Detected Language | English |
| Type | Student thesis, info:eu-repo/semantics/bachelorThesis, text |
| Format | application/pdf |
| Rights | info:eu-repo/semantics/openAccess |
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