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| ???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
|---|---|---|
| dc.contributor.advisor | 陳志成(Chih-Cheng Chen) | |
| dc.contributor.author | Hung-Chin Wang | en |
| dc.contributor.author | 王宏晉 | zh_TW |
| dc.date.accessioned | 2021-06-08T06:02:15Z | - |
| dc.date.copyright | 2007-07-30 | |
| dc.date.issued | 2007 | |
| dc.date.submitted | 2007-07-25 | |
| dc.identifier.citation | Akopian AN, Chen CC, Ding Y, Cesare P, Wood JN (2000) A new member of the acid-sensing ion channel family. Neuroreport 11:2217-2222.
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| dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/25098 | - |
| dc.description.abstract | 在研究單一細胞分子的特性上,利用單一細胞層級反轉錄多鏈鎖反應是一個非常有效力的技術。利用此技術，在同一顆細胞中可以了解很多基因的表現，如此可以更了解單一細胞的特性。對於一些特異性非常高的細胞，如:背根神經節神經細胞，因為它們具有很複雜的功能及特性，因此要去區分它們的細胞種類是非常困難的。所以若要了解它們的特性及種類，需要更多的分子訊息，而單一細胞層級反轉錄多鏈鎖反應就為一個好的技術。可是此技術仍有很多需要改進的空間，在這個研究中，我嘗試去改善此技術包含在收集細胞的方法、多鏈鎖反應的敏感度及降低操作過程中可能造成的汙染，進而去提高此技術的效力，可以在一個細胞中偵測超過100個基因的表現。藉由改善此技術，我試圖去研究一群表現第三型酸離子通道 (ASIC3) 肌肉訊息傳入神經元的分子特性。結果發現，與其他不表現ASIC3的肌肉訊息傳入神經元比較，有表現ASIC3的肌肉訊息傳入神經元具有非常不同的分子特性。在此研究中，我成功地建立一個適當且有效力的技術去探討一群具有高特異性的神經細胞的分子特性。 | zh_TW |
| dc.description.abstract | Single cell RT-PCR is a powerful tool to explore the molecular identity of single cells. It can determine the expression of many genes in a single cell simultaneously and help to characterize the diverse cell population like dorsal root ganglion DRG neurons. However, there are still some limitations of single cell RT-PCR to obtain a large amount of informative data in gene expression from a single cell. Therefore, I established a new protocol to perform single cell RT-PCR with modification on cell harvesting approach, increased sensitivity of gene determination and reduced contamination. Ideally, this method will be able to detect over 100 genes in a single neuron. I applied this modified single cell RT-PCR to investigate the molecular identity of muscle afferent neurons and showed that ASIC3 expressing muscle afferent neurons had distinct molecular identity from other neurons. The single cell RT-PCR data were consistent with previous studies using immunostaining. In the thesis, I successfully established a powerful technique to investigate the molecular identity of a diverse neuronal population. | en |
| dc.description.provenance | Made available in DSpace on 2021-06-08T06:02:15Z (GMT). No. of bitstreams: 1 ntu-96-R94b41010-1.pdf: 4476933 bytes, checksum: 1de18bfcff2d892af9ea526ba0df9e89 (MD5) Previous issue date: 2007 | en |
| dc.description.tableofcontents | Contents
口試委員審定 書……………………………………………………………………i Acknowledgement…………………………………………………ii 摘要………………………………………………………………iii Abstract…………………………………………………………iv Contents……………………………………………………v~viii List of figures…………………………………………ix~xi List of tables……………………………………………xii Chapter 1 Introduction……………………………………………………1 1.1 Single cell RT-PCR…………………………………………………………2-3 1.2 Why Single cell RT-PCR?..............................................4-5 1.3 Acid sensing ion channel 3 (ASIC3) and acidosis induce muscle pain……5-7 1.4 The objective of the thesis……………………………………………………7-8 Chapter 2 Material and Method…………………………………………………9 2.1 Muscle afferent neurons………………………………………………………9 2.2 Primary cell culture………………………………………………………10-11 2.3 Cell harvesting…………………………………………11 2.4 Single cell RNA purification…………………………………………………12 2.5 Reverse transcription…………………………………………………………12 2.6 Multiplex nested primer set…………………………………………………..13 2.7 Single cell multiplex-nested PCR…………………………………………13-14 2.8 Statistic analysis of data………………………………………………………14 Chapter 3 Results……………………………………………………………….15 3.1 Sensitivity of single cell RT-PCR…………………………………………16-18 3.1.1 Session of cell harvesting approach………………………………………..18 3.1.1.1 Preliminary works on cell harvesting and problems………………….18-19 3.1.1.2 Problems of cell harvesting approach…………………………………….20 3.1.1.3 Developing better cell harvesting approach in our system……………20-22 3.1.2 Session of RNA isolation and reverse transcription……………………22-23 3.1.2.1 RNA isolation and modification……………………………………..23-24 3.1.2.2 Reverse transcription.……………………………………………….24-25 3.1.3 Session of multiplex and nested PCR…………………………………….25 3.1.3.1 Multiplex PCR……………………………………………………….26-27 3.1.3.2 Why performed nested PCR…………………………………………27-28 3.1.3.3 Optimization of multiplex PCR condition……………………………28-29 3.1.3.4 Multiplex nested PCR sensitivity in a single neuron…………………29-30 3.1.3.5 Template effects and other interference in multiplex PCR……………30-31 3.2 Contamination issue of single cell RT-PCR………………………………...32 3.2.1 Reducing cross contaminations between cells…………………………..32-33 3.2.2 Reducing PCR product contamination……………………………………...33 3.2.2.1 Reducing contamination by ultrapure filter tips…………………………..34 3.2.2.2 Separate places for RT-PCR and PCR products analysis…………………34 3.2.3 Distinguishing genomic DNA contamination by intron spanning primers……………………………………………………………35 3.2.4 Careful handing to avoid contamination………………………………..36-37 3.2.5 Monitoring contamination when performing single cell RT-PCR………37-38 3.3 Exploring the molecular identity of muscle nociceptors by single cell RT-PCR…………………………………………………….39-40 3.3.1 Characterization of muscle afferent neurons………………………………..40 3.3.2 Establishment of multiplex nested primer set for exploring muscle afferent neurons………………………………………………….40-42 3.3.3 Gene expression determination by multiplex nested PCR………………….42 3.3.3.1 Threshold for detection of genes………………………………………42-43 3.3.3.2 Criteria for informative neurons…………………………………………..43 3.3.4 Distinct molecular identity of ASIC3 expressing muscle afferent neurons……………………………………………………44 3.3.5 Correlation of genes with functions……………………………………44-46 3.3.6 Fidelity of single cell RT-PCR…………………………………………….46 3.4 Summary…………………………………………………………………….47 Chapter 4 Discussion……………………………………….………………….48 4.1 Limitation of single cell RT-PCR and data interpretation…………………49-51 4.2 Distinct gene expression of ASIC3 expressing muscle afferent neurons….51-53 4.3 Fidelity of single cell RT-PCR……………………………………………..53-55 4.4 Results comparing with previous immunostaining studies………………..55-56 Reference……………………………………………………………………...57-63 List of Figure Figure 1 One copy gene can be detected after 35 cycle amplification by PCR ideally………………………………………………………….64 Figure 2 PCR amplification………………………………………………………65 Figure 3 Harvesting single DRG neuron by two pipette approach………………66 Figure 4 Cell harvesting by single pipette approach……………………………..67 Figure 5 Single neuron without detection of NTFRs also showed no detection of other genes……………………………………………68 Figure 6 Surface tension and waterflow of external solution would interfere with cell holding when cell was harvested by single pipette approach………………………………………………..69 Figure 7 Improved quality of modified cell harvesting approach………………70 Figure 8 Fluorogold injection into gastrocnemius muscle for tracing muscle afferent DRG neurons…………………………………………71 Figure 9 Cell storing in lysis buffer for further RNA isolation and RNA purification protocol…………………………………………….72 Figure 10 Demonstrating successful reverse transcription for mRNA with long length of last exon…………………………………………73 Figure 11 Flowchart of multiplex–nested PCR………………………………….74 Figure 12 Nested primer increased the efficiency and accuracy of two steps PCR………………………………………………………..75 Figure 13 Optimization of concentration of primers for multiplex PCR………………………………………………………………….76 Figure 14 Genomic DNA detection in a single neuron showed that sensitivity of multiplex-nested PCR was one copy………………….77 Figure 15 Interference between genes when multiplex PCR was Performed……………………………………………………………78 Figure 16 Reducing contaminations of single cell RT-PCR……………………79 Figure 17 Intron spanning primers could distinguish genomic DNA from mRNA…………………………………………………………80 Figure 18 Contaminations from non-pure double distilled water……………..81 Figure 19 Careful handing of PCR to avoid contaminations between samples……………………………………………………………..82 Figure 20 Negative controls of multiplex nested PCR………………………..83 Figure 21 Negative controls were performed to secure the gene expression of single neuron………………………………………..84 Figure 22 Multiplex nested primer sets for exploring the molecular identity of single neuron…………………………………………..85 Figure 23 The flowchart of multiplex nested primer set establishment………86 Figure 24 Summary of steps for exploring gene expression in a single neuron………………………………………………….87 Figure 25 SA inhibition was correlated with expression of ASICs subtypes in muscle afferent neurons………………………………88 Figure 26 Different current kinetics of ASIC3 only and ASIC3, TRPV1 (+) muscle afferent neurons………………………………89 Figure 27 Correlation of gene and cell size in muscle afferent neurons…….90 Figure 28 Standard protocol of modified single cell RT-PCR for investigating molecular identity of muscle afferent neurons……91 List of Table Table 1 Improvement of cell harvesting quality by two pipette approach………………………………………………………………92 Table 2 Determining gene expression in single neurons without DNase I treatment…………………………………………………….93 Table 4 Non-detection of ASIC3 in ASIC3-/- neurons with transient H+-gated current. …………………………………………………….94 Table 5 Comparison of gene expression profile between ASIC3 expressing and non-ASIC3 muscle afferent neurons………………..95 Table 6 Correlation of cell size, membrane potential, AP configurations and genes……………………………………………96 Table 7 Non-bias of data selection after performing single cell RT-PCR…………………………………………………………97 Table 8 Primer list of single cell RT-PCR………………………………98-101 | |
| dc.language.iso | en | |
| dc.subject | 肌肉訊息傳入神經元 | zh_TW |
| dc.subject | 單一細胞層級反轉錄多鏈鎖反應 | zh_TW |
| dc.subject | 特異性 | zh_TW |
| dc.subject | 背根神經節 | zh_TW |
| dc.subject | 第三型酸離子通道 | zh_TW |
| dc.subject | diverse | en |
| dc.subject | muscle afferent neuron | en |
| dc.subject | ASIC3 | en |
| dc.subject | dorsal root ganglion | en |
| dc.subject | single cell RT-PCR | en |
| dc.title | 利用改良式單一細胞層級反轉錄多聚酶鏈鎖
反應技術去探討肌肉痛覺神經元的分子特性 | zh_TW |
| dc.title | Improved single cell RT-PCR for investigating
molecular identity of muscle nociceptor | en |
| dc.type | Thesis | |
| dc.date.schoolyear | 95-2 | |
| dc.description.degree | 碩士 | |
| dc.contributor.coadvisor | 閔明源(Ming-Yuan Min) | |
| dc.contributor.oralexamcommittee | 孫維欣,黃翊恭 | |
| dc.subject.keyword | 單一細胞層級反轉錄多鏈鎖反應,特異性,背根神經節,第三型酸離子通道,肌肉訊息傳入神經元, | zh_TW |
| dc.subject.keyword | single cell RT-PCR,diverse,dorsal root ganglion,ASIC3,muscle afferent neuron, | en |
| dc.relation.page | 101 | |
| dc.rights.note | 未授權 | |
| dc.date.accepted | 2007-07-27 | |
| dc.contributor.author-college | 生命科學院 | zh_TW |
| dc.contributor.author-dept | 動物學研究研究所 | zh_TW |
| Appears in Collections: | 動物學研究所 | |
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