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研究生(外文):Peu-Chen Huang
論文名稱(外文):Establishment of a Multi-sampling Reporter System to Monitor Activities of microRNAs
指導教授(外文):Lo-Chun Au
外文關鍵詞:reporter systemmulti-samplingsecretable enzymesmiRNA activitymedium
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目前有許多方法可用於定量miRNA或偵測miRNA的活性。而北方墨點法(Northern blot)、即時定量RT-PCR、生物晶片(microarray)和螢火蟲螢光酵素播報系統(firefly luciferase reporter system)則是最廣為使用的幾種方法。然而這些方法都需要將待測細胞溶解後方可進行分析,因此無法持續監控同一群細胞在不同時間點的miRNA表現。在以上述方法進行不同時間點的miRNA偵測時必須要培養許多盤細胞,而此舉將增將實驗的複雜性,且細胞盤與盤間的差異可能會對實驗的精確度造成影響。有鑑於此,在本實驗中我們利用兩種可由細胞分泌出來的生物冷光酵素,一個用於偵測miRNA而另一個則用於監控轉染的效率,成功的建立一個可重複取樣的播報系統。由於該系統只需收取少量的細胞培養液即可進行分析,因此能在長時間中對同一群細胞進行重複取樣並偵測該細胞在不同時間點miRNA的活性。而在本實驗中我們也對該系統的特性、可行性與優點進行深入的探討。
There are several methods to detect the relative levels or activities of miRNAs including Northern blot analysis, quantitative real-time RT-PCR, microarray and firefly luciferase reporter system. However, lysis of tested cells is necessary for all of these methods. Therefore, we cannot use these methods to continuously monitor a miRNA in the same population of cells, and need to prepare several sets of wells/dishes of cells for tracking the alteration of a miRNA level in the time-course studies. It is uneconomical and experimental accuracy may be compromised by the variations between samples.
In this study, we developed a multi-sampling reporter system by using two secretable bioluminescence-generating enzymes ─ Metrida luciferase and secreted alkaline phosphatase. One is for a reporter and the other for an internal control. Therefore we can monitor miRNA activities in the same population of cells over time simply by withdrawing aliquots of the culture medium followed by assays. We also demonstrated characteristic, practicability and benefits of this system are in this report.
論文電子檔著作權授權書 i
論文審定同意書 ii
誌謝 iii
中文摘要 iv
Abstract v
Table of Contents vi
List of Figures viii
Chapter 1: Introduction 1
1.1 An overview of microRNAs 1
1.2 Quantification of miRNAs 3
1.3 The secretable reporting enzymes 6
1.4 miR-15/16 family 8
1.5 Objectives 10
Chapter 2: Materials and Methods 12
2.1 Cell lines and cultures 12
2.2 The plasmids for the multi-sampling reporter system 12
2.3 Assay of MLuc activity 14
2.4 Assay of SEAP activity 15
2.5 Transfection of HEK293T 16
2.6 Cell lysis 16
2.7 Detection of secretable MLuc by X-ray films 16
2.8 Detection of level change of endogenous miR-16 17
2.9 Statistical analysis 18
Chapter 3: Results 19
3.1 The plasmids for multi-sampling reporter system 19
A. Screening of pMLuc-UTR by PCR 19
B. Sequencing result of pMLuc-UTR 20
3.2 Characterization of the multi-sampling reporter system 20
A. Detection of secretable MLuc by X-ray films 21
B. Detection of the reporting enzymes in media and lysates 21
C. Comparison between variations in commercial assays and modified assays 21
D. Comparison with the results made with different reagents 22
E. Background signals of MLuc and SEAP in culture media 23
F. Stability of MLuc and SEAP after freeze 23
G. Stability of MLuc and SEAP in culture media 23
H. Effects of dilution on repression folds 23
3.3 Reporting ability of the multi-sampling reporter system 24
A. Responsibility of the multi-sampling reporter system to pre-miR-16 24
B. Reporting ability of multi-sampling 24
C. Sensitivity of the multi-sampling reporting system 25
D. Detection of level change of endogenous miR-16 26
Chapter 4: Conclusion and Discussion 27
References 45
Appendix 48
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