| Summary: | 碩士 === 國立臺灣大學 === 應用力學研究所 === 106 === Liquid biopsy has been applied to clinical research and disease management in recent years. It provides a non-invasive, low risk and time saving approach to access bio-samples such as RNA, DNA, proteins, exosomes, and cells from body fluids. Given that nucleic acid is the origin of abnormal mutants, detecting RNA might be able to collect information before other biomarkers show disease signal which might be suitable for early disease detection.
Enrichment of RNA is an important process to characterize the nucleic acid. Although there are many RNA isolation kits on the market, most of them are manually operated, which is time-consuming and operator dependent. Only a few methods are automated, and they are operated by high-throughput, large-volume devices.
This thesis aims to developing an automated RNA enrichment method and a microfluidic assay. Considering the automation of a centrifuge and input of reagents, a disk-based RNA isolation was designed. The protocol modified from CatchGeneTM Cell/Exosome miRNA Kit was applied to an acrylic disk with chambers and microfluidic channels motivated by the ease of fluid manipulation via centrifugation. First, the mixing ability of the disk was tested and compared with that from standard kit protocol.
Then, the stability and the quality of the RNA enriched by the disk protocol were validated using MCF7 cells as samples. Results of electrophoresis showed that the RNA samples enriched by three identical disks had the same quality as tube protocol, which suggest the performance of the system was stable. Results detected by NanodropTM and QubitTM showed that the performance of the disk was stable since the coefficient of variation of the RNA concentration was only 0.18 and the quantity of total RNA was as high as 75.5% of that enriched by standard kit protocol with only 13% standard deviation. The quality of the RNA sample was evaluated by the similarity between positive control and disk outcome, which is the similarity of the genetic expression proportion of four biomarkers: RPP30, ER, HER2 and Ki67. Overall, the proportion of genetic expression level of RNA sample enriched by the disk was very similar to that enriched by the standard protocol.
Besides MCF7 cells, other cells such as BT474 and WBC were also used to validate the application of the microfluidic platform to different samples. Results of electrophoresis and qPCR both showed high quality. The genetic expression proportion of RNA sample enriched from BT474 and WBC in two methods showed high similarity.
After validation using cells, exosomes from plasma of healthy donor were introduced to the system. Results detected by qPCR with three biomarkers, miR21, let-7 and GAPDH, showed that the microfluidic platform was able to enrich exosomal RNA with high sensitivity.
In conclusion, the microfluidic platform was able to enrich extracellular and exosomal RNA with high quality and stability. System automation was the motivation may enable further utilization of the microfluidic disk-based technology.
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