微流離心碟盤平台應用於細胞與外泌體之核醣核酸萃取之研究

Abstract

近年來，液體活檢已被應用於許多臨床研究和病程監測之中。比起需要手術的組織活檢，液體活檢的優點在於非侵入式，低風險，便利省時且操作門檻低 。從液體活檢中，藉由鎖定分析特定的生物標記，像是去氧核醣核酸，核糖核酸，蛋白質，循環細胞或是外泌體，來得到與疾病相關的資訊。已知核糖核酸是影響體內蛋白變異的起源，若能夠檢測核糖核酸的表現變化，便有機會在其他生物標記尚未產生變異之前，在第一時間獲取病變的訊息。因此，富集核糖核酸的技術是極為關鍵的。目前市面上的富集方法，大部分是手動操作為主，雖然效果卓越，卻必須佔用操作者的雙手一段時間，生產的結果也因每次操作而有差異。能夠自動執行的富集方法極少且儀器本身體積龐大。 本研究致力於開發一套自動化核糖核酸富集系統與方法 。考慮到富集方法所需的離心力和試劑混合等自動化的建置，微流碟盤平台是最適合的。參考市面上不同的核糖核酸試劑組之後，採用開啟基因的細胞/外泌體小片段核糖核酸富集試劑組作為開發基礎，修改流程，在微流碟盤平台上完成同樣的效果，並測試試劑混合的效果和液體性質 。 碟盤的原型完成後，核酸富集能力的穩定性和品質都需要測試跟驗證。MCF7細胞株被選為測試檢體，除了Nanodrop和Qubit兩種方法檢測之外，也經過膠電泳的分析。膠電泳的結果顯示，由三片碟盤所富集的核酸都有著和由原試劑組所富集的核酸同樣的高品質，表示富集能力很穩定。同時，由兩種方法檢測出來核酸濃度，其變異係數只有0.18，表示濃度變動不大，很穩定，加上碟盤富集的核酸之濃度和試劑組所富集的結果相比，能達到試劑組75.5%的濃度，標準差為13%，表示不只產出穩定，品質高，也不會有太多的核酸損失。產出品質的測試方法還有一個，是分析核酸中不同的生物標記的量比例，藉此了解碟盤富集的核酸是否和試劑組富集的核酸擁有同樣的基因比例，以是否只有特定的基因被富集來判斷核酸品質的高低。使用即時聚合酶鏈式反應來檢測富集後的核酸中特定的生物標記有：RPP30，ER，HER2和Ki67。結果顯示，從三片碟盤的綜合分析來看，碟盤富集的核酸的基因組成和試劑組富集的相似度很高，核酸品質優良。 此外，為了測試微流碟盤平台對於不同樣本來源的富集能力，使用BT474細胞株，MCF7細胞株和白血球進行實驗。從膠電泳的結果可知，富集的核酸品質和由試劑組富集出來的一樣好。從即時聚合酶鏈式反應的結果可知，雖然碟盤富集的MCF7細胞株的核酸基因組成和試劑組富集的有差異，但另外兩組的結果都顯示高度相似，整體來說，核酸品質仍然是良好的。最後，外泌體作為液體活檢的高價值目標物，進入富集核酸的驗證 。不同於細胞的測試，使用let-7，miR-21和GAPDH作為生物標記去檢測，以相對基因表現量來做比較，結果顯示，本系統在富集外泌體的核糖核酸上有高靈敏性，在一定範圍的樣本體積量之內，都能夠富集出相對應的核糖核酸量。 總結來說，本研究開發出一套效能穩定的微流碟盤平台，能從細胞或外泌體樣本富集高品質的核糖核酸 。本系統的自動化將可能作為微流碟盤技術在核酸富集的應用之一。

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.