部分磷酸根甲基化之反義去氧核醣核酸探針與 微小核糖核酸雜交靈敏度與專一性之研究

以作者查詢圖書館館藏

、以作者查詢臺灣博碩士

、以作者查詢全國書目

、勘誤回報

、線上人數：44

、訪客IP：3.136.25.249

姓名

張晴雯(Ching-Wen Chang) 查詢紙本館藏

畢業系所

化學工程與材料工程學系

論文名稱

部分磷酸根甲基化之反義去氧核醣核酸探針與微小核糖核酸雜交靈敏度與專一性之研究
(Sensitive and specific microRNA hybridization using partially methylated phosphotriester antisense DNA probes)

相關論文

★ 類澱粉胜肽聚集行為之電腦模擬	★ 溶解度參數計算及量測於HPLC純化胜肽程序之最佳化研究
★ 利用恆溫滴定微卡計量測蛋白質分子於溶液中之第二維里係數與自我聚集之行為	★ 利用SPRi探討中性DNA探針相較於一般DNA探針在低鹽雜交環境下之優勢
★ 矽奈米線場效電晶體多點之核酸檢測研究	★ 使用不帶電中性核酸探針於矽奈米線場效電晶體檢測去氧核醣核酸與微核醣核酸之研究
★ 運用nDNA 修飾引子於PCR及qPCR平台以提升專一性之研究	★ 設計中性DNA引子及探針以提升PCR與qPCR專一性之研究
★ 使用中性不帶電去氧核醣核酸探針於矽奈米線場效電晶體檢測微核醣核酸之研究	★ 使用不帶電中性核酸探針於原位雜交技術檢測微核醣核酸之研究
★ 設計不帶電中性核酸探針於矽奈米線場效電晶體來改善富含GC鹼基核醣核酸之檢測專一性	★ 合成5’-MeNPOC-2’-deoxynucleoside p-methoxy phosphoramidite以作為應用於原位合成之新穎性中性核苷酸之研究
★ 立體紙基外泌體核酸萃取裝置應用於檢測不同微環境下癌細胞所釋放之外泌體與外泌體微小核醣核酸之表現量	★ 利用抗原結合區段之抗體片段探針於矽奈米線場效電晶體來改善抗原檢測濃度極限之研究
★ 利用表面電漿共振影像儀驗證最適化之抗非專一性吸附場效電晶體表面於血清環境下之免疫測定	★ 使用混合自組裝單層膜於矽奈米線場效電晶體檢測微小核醣核酸之研究

檔案

[Endnote RIS 格式]

[Bibtex 格式]

[相關文章]

[文章引用]

[完整記錄]

[館藏目錄]

[檢視]

[下載]

本電子論文使用權限為同意立即開放。
已達開放權限電子全文僅授權使用者為學術研究之目的，進行個人非營利性質之檢索、閱讀、列印。
請遵守中華民國著作權法之相關規定，切勿任意重製、散佈、改作、轉貼、播送，以免觸法。

摘要(中)

MicroRNAs (miRNAs)為一種非編碼的小片段RNA，在調節基因表達中發揮著重要的作用。然而，目前使用反義寡核苷酸與目標miRNA雜交作為檢測或抑制miRNA的方法，而這些方法包含原位雜交技術(ISH)、RT-qPCR和miRNA微陣列分析技術，但上述這些方法皆缺乏雜交的專一性及親和力。因此為了改善這些問題，我們在核酸序列探針上修飾中性去氧核醣核酸 (nDNA)，nDNA為一種人工DNA類似物，其磷酸骨幹經過特定位置的甲基化改質後，使帶負電的磷酸根轉變成磷酸三酯鍵而形成電中性，其可減少核酸雙股同時帶負電造成的靜電排斥效應，因此具有較佳的雜交穩定性。
本研究表明，藉由RT-qPCR檢測結果所示，部分甲基化的反義nDNA探針(N4 nDNA，具有四個nDNA修飾)可比DNA 探針更有效地抑制在人體血漿培養基中的miR-21進行逆轉錄反應(N4 nDNA探針具有較高的Ct值)。接著，我們使用具有miR-21表達的人類結腸癌細胞(HCT116)進行原位雜交技術(ISH)的檢測，使用N4 nDNA探針檢測HCT116細胞中的miR-21，可顯示出比使用一般DNA探針檢測到更高的染色訊號強度且具有檢測專一性。同樣地，藉由酵素結合免疫吸附分析法(ELISA)表明N4 nDNA探針與miRNA雜交的效率高於DNA探針。此外，N4 nDNA探針在細胞中不會引起免疫反應(與國家衛生研究院紀雅惠博士合作)，並且N4 nDNA探針在體外無細胞蛋白質合成(CFPS)系統中可抑制GFP mRNA進行轉譯(與日本東京工業大學地球生命研究所的Kosuke Fujishima教授合作)。
由於N4 nDNA探針對於DNase I具有抵抗能力並且不會在細胞中引發強烈的免疫反應，因此未來在開發nDNA探針作為調節體內基因表達的治療性核酸藥物是具有可能性的。

摘要(英)

MicroRNAs (miRNAs), a class of short non-coding RNA, play important roles in regulating gene expression. However, current miRNA hybridization methods for miRNA detection/inhibition using antisense oligonucleotides, including in situ hybridization (ISH), RT-qPCR, and miRNA microarray profiling technology, are lacking hybridization specificity and affinity. As such, to ameliorate these problems, the neutralized DNA, an emerging class of DNA oligonucleotides chemically synthesized with site-specific internucleoside methyl phosphotriester linkages was used as a hybridization probe instead of DNA. The reduction of inter-strand charge repulsion of nucleotide duplexes results in stronger binding between nDNA and other nucleic acids.
This study suggested that partially methylated antisense nDNA (N4 nDNA, with four modifications) probes inhibited reverse transcription of miR-21 more efficiently (higher Ct value) than DNA probes in the human plasma-like medium, as demonstrated by the RT-qPCR assays. Subsequently, we performed in situ hybridization analysis using a miR-21-expressing colorectal cancer cell line (HCT116). HCT116 cells stained with N4 nDNA antisense probes revealed a greater detection intensity and specificity than cells stained with DNA probes. Consistently, enzyme-linked immunosorbent assays (ELISA) suggested that the miRNA hybridization efficiency of N4 nDNA antisense probes was greater than that of DNA probes. In addition, the cell-based immune response of the N4 nDNA antisense probe was immune-negative (cooperated with Dr. Ya-Hui Chi at National Health Research Institutes) and the N4 nDNA antisense prob was also inhibited in vitro GFP mRNA translation (cooperated with Prof. Kosuke Fujishima at Earth-Life Science Institute).
As N4 nDNA probes are resistant to DNase I and do not elicit a strong immune response in cells, future development of nDNA probes as therapeutic nucleic acid drugs for in vivo gene expression modulation is possible.

關鍵字(中)

★ 合成寡核苷酸
★ 核酸類似物
★ 定量即時聚合酶連鎖反應
★ 酵素結合免疫吸附分析法
★ 微核糖核酸
★ 直腸結腸癌

關鍵字(英)

★ Synthesized oligonucleotides
★ Nucleic acid analogues
★ qPCR
★ ELISA
★ microRNA
★ colorectal cancer

論文目次

摘要.......................................................i
ABSTRACT.................................................iii
致謝.......................................................v
目錄.....................................................vii
圖目錄....................................................xi
表目錄...................................................xiv
一、緒論...................................................1
二、文獻回顧................................................3
2.1核酸分子介紹.............................................3
2.1.1核酸分子..............................................3
2.1.2去氧核醣核酸(Deoxyribonucleic acid, DNA)...............4
2.1.3核醣核酸(Ribonucleic acid, RNA).......................5
2.1.4微小核醣核酸(microRNA).................................7
2.2核酸類似物..............................................9
2.2.1鎖核酸(Lock nucleic acid, LNA)........................9
2.2.2肽核酸(Peptide nucleic acid, PNA)....................11
2.2.3中性去氧核醣核酸(Neutralized DNA, nDNA)...............12
2.3反義寡核苷酸對基因的調控.................................18
2.4基因分子檢測............................................20
2.4.1聚合酶鏈鎖反應(Polymerase chain reaction, PCR)........20
2.4.2即時定量聚合酶鏈鎖反應(Quantitative real-time polymerase chain reaction, qPCR).....................................23
2.5原位雜交技術(in situ hybridization, ISH)................26
2.5.1原位雜交技術的基本原理與發展歷程.......................26
2.5.2原位雜交技術之訊號放大.................................29
2.6酵素結合免疫吸附分析法(Enzyme-linked immunosorbent assay, ELISA)....................................................32
2.7無細胞蛋白質合成(Cell-free protein synthesis system, CFPS)系統......................................................34
三、實驗方法與儀器設備......................................36
3.1實驗藥品...............................................36
3.1.1細胞培養.............................................36
3.1.2序列設計.............................................36
3.1.3核酸探針3′端DIG合成...................................37
3.1.4原位雜交技術(in situ hybridization, ISH)..............37
3.1.5模擬生物體內miRNA雜交.................................38
3.1.6細胞轉染.............................................38
3.1.7核酸萃取.............................................38
3.1.8即時聚合酶鏈式反應....................................38
3.1.9洋菜凝膠電泳(Agarose gel).............................39
3.2儀器設備...............................................40
3.3實驗方法...............................................41
3.3.1細胞解凍.............................................41
3.3.2細胞冷凍保存..........................................42
3.3.3細胞培養.............................................43
3.3.4核酸探針3′端DIG合成...................................44
3.3.5原位雜交技術 (in situ hybridization, ISH).............45
3.3.6模擬生物體內miRNA雜交.................................47
3.3.7細胞轉染.............................................48
3.3.8微小核醣核酸萃取......................................49
3.3.9逆轉錄及時聚合酶鍊式反應(qRT-PCR)......................51
3.3.10酵素結合免疫吸附分析法(Enzyme-linked immunosorbent assay, ELISA).............................................55
3.3.11洋菜凝膠電泳(Agarose gel)............................57
四、實驗結果與討論.........................................58
4.1建立反義寡核苷酸探針在Human plasma-like medium中抑制miRNA的逆轉錄....................................................58
4.1.1不同修飾數目nDNA探針對miR-21逆轉錄的抑制能力............65
4.1.2nDNA探針對DNase I的抵抗能力...........................68
4.1.3nDNA探針在不同逆轉錄時間的抑制能力.....................70
4.1.4不同nDNA探針濃度對miRNA逆轉錄的抑制能力................72
4.2通過N4 nDNA探針在癌細胞HCT116中定量miR-21................74
4.2.1使用原位雜交技術通過N4 nDNA探針定量miR-21..............75
4.2.2使用ELISA通過N4 nDNA探針定量miR-21....................81
4.3利用nDNA探針在人類結腸癌細胞HCT116中抑制miR-21...........85
五、結論..................................................87
六、未來展望...............................................89
七、參考文獻...............................................90
八、附錄..................................................96
8.1使用利用高效液相層析法(High performance liquid chromatography, HPLC)分析DNA及N4 nDNA產物.................96
8.2不同探針在加熱/未加熱DNase I之p-value....................97
8.3通過N4 nDNA探針在無細胞蛋白質合成系統中抑制GFP mRNA.......97
8.4不同去除保護基試劑對於合成nDNA序列分子量的影響............99

參考文獻

[1] R. Dahm, "Discovering DNA: Friedrich Miescher and the early years of nucleic acid research," Human Genetics, vol. 122, no. 6, pp. 565-581, Jan 2008.
[2] J. D. Watson and F. H. C. Crick, "MOLECULAR-STRUCTURE OF NUCLEIC-ACIDS - A STRUCTURE FOR DEOXYRIBOSE NUCLEIC-ACID," Jama-Journal of the American Medical Association, vol. 269, no. 15, pp. 1966-1967, Apr 1993.
[3] A. Leslie, S. Arnott, R. Chandrasekaran, and R. Ratliff, "Polymorphism of DNA double helices," Journal of molecular biology, vol. 143, no. 1, pp. 49-72, 1980.
[4] J. Fohrer, M. Hennig, and T. Carlomagno, "Influence of the 2′-hydroxyl group conformation on the stability of A-form helices in RNA," Journal of molecular biology, vol. 356, no. 2, pp. 280-287, 2006.
[5] P. G. Higgs, "RNA secondary structure: physical and computational aspects," Quarterly reviews of biophysics, vol. 33, no. 3, pp. 199-253, 2000.
[6] B. Wightman, I. Ha, and G. Ruvkun, "Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans," Cell, vol. 75, no. 5, pp. 855-862, 1993.
[7] R. C. Lee, R. L. Feinbaum, and V. Ambros, "The C. elegans heterochronic gene lin-4 encodes small RNAs with antisense complementarity to lin-14," cell, vol. 75, no. 5, pp. 843-854, 1993.
[8] L.-A. MacFarlane and P. R Murphy, "MicroRNA: biogenesis, function and role in cancer," Current genomics, vol. 11, no. 7, pp. 537-561, 2010.
[9] G. Hutvagner and P. D. Zamore, "A microRNA in a multiple-turnover RNAi enzyme complex," Science, vol. 297, no. 5589, pp. 2056-2060, 2002.
[10] M. Shah, A. Ferrajoli, A. Sood, G. Lopez-Berestein, and G. Calin, "microRNA therapeutics in cancer—An emerging concept. eBioMedicine. 2016; 12: 34-42," ed: DOI.
[11] K. B. Reddy, "MicroRNA (miRNA) in cancer," Cancer Cell International, vol. 15, no. 1, pp. 1-6, 2015.
[12] B. Zhang, X. Pan, G. P. Cobb, and T. A. Anderson, "MicroRNAs as oncogenes and tumor suppressors," Developmental Biology, vol. 302, no. 1, pp. 1-12, 2007.
[13] S. Lin and R. I. Gregory, "MicroRNA biogenesis pathways in cancer," Nature reviews cancer, vol. 15, no. 6, pp. 321-333, 2015.
[14] J. Zhao, Y. S. Zhang, and G. H. Zhao, "Emerging role of microRNA-21 in colorectal cancer," Cancer Biomarkers, vol. 15, no. 3, pp. 219-226, 2015.
[15] S. K. Singh, A. A. Koshkin, J. Wengel, and P. Nielsen, "LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition," Chemical communications, no. 4, pp. 455-456, 1998.
[16] J. Wengel et al., "LNA (locked nucleic acid) and the diastereoisomeric α-L-LNA: conformational tuning and high-affinity recognition of DNA/RNA targets," Nucleosides, Nucleotides and Nucleic Acids, vol. 20, no. 4-7, pp. 389-396, 2001.
[17] S. Obika et al., "Synthesis of 2′-O, 4′-C-methyleneuridine and-cytidine. Novel bicyclic nucleosides having a fixed C3,-endo sugar puckering," Tetrahedron Letters, vol. 38, no. 50, pp. 8735-8738, 1997.
[18] K. Fakhfakh, O. Marais, X. B. J. Cheng, J. R. Castañeda, C. B. Hughesman, and C. Haynes, "Molecular thermodynamics of LNA: LNA base pairs and the hyperstabilizing effect of 5′‐proximal LNA: DNA base pairs," AIChE Journal, vol. 61, no. 9, pp. 2711-2731, 2015.
[19] M. Petersen and J. Wengel, "LNA: a versatile tool for therapeutics and genomics," Trends in biotechnology, vol. 21, no. 2, pp. 74-81, 2003.
[20] Y. You, B. G. Moreira, M. A. Behlke, and R. Owczarzy, "Design of LNA probes that improve mismatch discrimination," Nucleic acids research, vol. 34, no. 8, pp. e60-e60, 2006.
[21] A. Válóczi, C. Hornyik, N. Varga, J. Burgyán, S. Kauppinen, and Z. Havelda, "Sensitive and specific detection of microRNAs by northern blot analysis using LNA-modified oligonucleotide probes," Nucleic acids research, vol. 32, no. 22, pp. e175-e175, 2004.
[22] M. Castoldi et al., "A sensitive array for microRNA expression profiling (miChip) based on locked nucleic acids (LNA)," Rna, vol. 12, no. 5, pp. 913-920, 2006.
[23] J. C. R. Politz, F. Zhang, and T. Pederson, "MicroRNA-206 colocalizes with ribosome-rich regions in both the nucleolus and cytoplasm of rat myogenic cells," Proceedings of the National Academy of Sciences, vol. 103, no. 50, pp. 18957-18962, 2006.
[24] E. Wienholds et al., "MicroRNA expression in zebrafish embryonic development," Science, vol. 309, no. 5732, pp. 310-311, 2005.
[25] T. Donnem et al., "Prognostic impact of MiR-155 in non-small cell lung cancer evaluated by in situ hybridization," Journal of translational medicine, vol. 9, no. 1, pp. 1-9, 2011.
[26] J. T. Pena et al., "MiRNA in situ hybridization in formaldehyde and EDC–fixed tissues," Nature Methods, vol. 6, no. 2, pp. 139-141, 2009.
[27] M. de Planell-Saguer and M. C. Rodicio, "Detection methods for microRNAs in clinic practice," Clinical biochemistry, vol. 46, no. 10-11, pp. 869-878, 2013.
[28] M. P. Johnson, L. M. Haupt, and L. R. Griffiths, "Locked nucleic acid (LNA) single nucleotide polymorphism (SNP) genotype analysis and validation using real‐time PCR," Nucleic acids research, vol. 32, no. 6, pp. e55-e55, 2004.
[29] U. A. Ørom, S. Kauppinen, and A. H. Lund, "LNA-modified oligonucleotides mediate specific inhibition of microRNA function," Gene, vol. 372, pp. 137-141, 2006.
[30] S. Tomac et al., "Ionic effects on the stability and conformation of peptide nucleic acid complexes," Journal of the American Chemical Society, vol. 118, no. 24, pp. 5544-5552, 1996.
[31] V. V. Demidov et al., "Stability of peptide nucleic acids in human serum and cellular extracts," Biochemical pharmacology, vol. 48, no. 6, pp. 1310-1313, 1994.
[32] S. Rigby et al., "Fluorescence in situ hybridization with peptide nucleic acid probes for rapid identification of Candida albicans directly from blood culture bottles," Journal of clinical microbiology, vol. 40, no. 6, pp. 2182-2186, 2002.
[33] E. Brognara et al., "Peptide nucleic acids targeting miR-221 modulate p27Kip1 expression in breast cancer MDA-MB-231 cells," International journal of oncology, vol. 41, no. 6, pp. 2119-2127, 2012.
[34] H. M. Moody, M. H. van Genderen, L. H. Koole, H. J. Kocken, E. M. Meijer, and H. M. Buck, "Regiospecific inhibition of DNA duplication by antisense phosphate-methylated oligodeoxynucleotides," Nucleic Acids Research, vol. 17, no. 12, pp. 4769-4782, 1989.
[35] M. H. van Genderen, L. H. Koole, and H. M. Buck, "Hybridization of phosphate‐methylated DNA and natural oligonucleotides. Implications for protein‐induced DNA duplex destabilization," Recueil des Travaux Chimiques des Pays‐Bas, vol. 108, no. 1, pp. 28-35, 1989.
[36] T. C. Kuo et al., "Reduction of interstrand charge repulsion of DNA duplexes by salts and by neutral phosphotriesters–contrary effects for harnessing duplex formation," Journal of the Taiwan Institute of Chemical Engineers, vol. 110, pp. 1-7, 2020.
[37] S. C. Chou, Y. J. Lai, X. z. Zhuo, W. Y. Chen, and S. Y. Li, "Increasing the λ-Red mediated gene deletion efficiency in Escherichia coli using methyl phosphotriester-modified DNA," Journal of the Taiwan Institute of Chemical Engineers, p. 104297, 2022.
[38] M. P. R. Paul S. Miller, * Akira Murakami, Kathleen R. Blake, Shwu-Bin Lin, and Cheryl H. Agris, "Solid-Phase Syntheses of Oligodeoxyribonucleoside Methylphosphonates1," Biochemistry, 1986.
[39] B. R. Meade et al., "Efficient delivery of RNAi prodrugs containing reversible charge-neutralizing phosphotriester backbone modifications," Nature Biotechnology, vol. 32, no. 12, pp. 1256-1261, 2014.
[40] H. M. Buck, "Phosphate-methylated oligonucleotides past, present and future," Journal of Biophysical Chemistry, vol. 11, no. 03, p. 27, 2020.
[41] H. M. Buck et al., "Phosphate-methylated DNA aimed at HIV-1 RNA loops and integrated DNA inhibits viral infectivity," Science, vol. 248, no. 4952, pp. 208-212, 1990.
[42] 林耀能 and Y.-n. Lin, "磷酸根甲基化去氧核醣核酸：合成、定性及其應用之研究;Phosphate-Methylated DNA as Neutralized DNA (nDNA)：Synthesis, Properties and Potential Applications," 2015.
[43] M. H. Caruthers, "Gene synthesis machines: DNA chemistry and its uses," Science, vol. 230, no. 4723, pp. 281-285, 1985.
[44] 陳奕儒 and Y.-j. Chen, "探討中性DNA與一般DNA雜交反應熱力學與結合機制之研究;Studies of thermodynamic and mechanism for neutralized DNA (nDNA)/DNA and DNA/DNA duplex formation," 2016.
[45] W.-P. Hu, C.-C. Tsai, Y.-S. Yang, H. W.-H. Chan, and W.-Y. Chen, "Synergetic improvements of sensitivity and specificity of nanowire field effect transistor gene chip by designing neutralized DNA as probe," Scientific reports, vol. 8, no. 1, pp. 1-8, 2018.
[46] W. Y. Chen, H. C. Chen, Y. S. Yang, C. J. Huang, H. W. H. Chan, and W. P. Hu, "Improved DNA detection by utilizing electrically neutral DNA probe in field-effect transistor measurements as evidenced by surface plasmon resonance imaging," Biosensors and Bioelectronics, vol. 41, pp. 795-801, 2013.
[47] W.-C. Chou, W.-P. Hu, Y.-S. Yang, H. W.-H. Chan, and W.-Y. Chen, "Neutralized chimeric DNA probe for the improvement of GC-rich RNA detection specificity on the nanowire field-effect transistor," Scientific reports, vol. 9, no. 1, pp. 1-10, 2019.
[48] T. L. Li et al., "Designed phosphate-methylated oligonucleotides as PCR primers for SNP discrimination," Analytical and Bioanalytical Chemistry, vol. 411, no. 17, pp. 3871-3880, 2019.
[49] T.-L. Li, "使用不帶電中性核酸探針於原位雜交技術檢測微核醣核酸之研究," National Central University, 2018.
[50] N. Dias and C. Stein, "Antisense oligonucleotides: basic concepts and mechanisms," Molecular cancer therapeutics, vol. 1, no. 5, pp. 347-355, 2002.
[51] Y. J. Tao et al., "Antisense oligonucleotides against microRNA-21 reduced the proliferation and migration of human colon carcinoma cells," Cancer Cell International, vol. 15, Aug 2015, Art. no. 77.
[52] R. Nedaeinia et al., "Locked nucleic acid anti-miR-21 inhibits cell growth and invasive behaviors of a colorectal adenocarcinoma cell line: LNA-anti-miR as a novel approach," Cancer Gene Therapy, vol. 23, no. 8, pp. 246-253, Aug 2016.
[53] J. Bartlett and D. Stirling, "A short history of the polymerase chain reaction," in PCR protocols: Springer, 2003, pp. 3-6.
[54] E. Van Rooij, "The art of microRNA research," Circulation research, vol. 108, no. 2, pp. 219-234, 2011.
[55] J. Wilhelm and A. Pingoud, "Real‐time polymerase chain reaction," Chembiochem, vol. 4, no. 11, pp. 1120-1128, 2003.
[56] J. N. Wilcox, "Fundamental principles of in situ hybridization," Journal of Histochemistry & Cytochemistry, vol. 41, no. 12, pp. 1725-1733, 1993.
[57] J. G. Gall, "The origin of in situ hybridization–a personal history," Methods, vol. 98, pp. 4-9, 2016.
[58] L. Arnould et al., "Agreement between chromogenic in situ hybridisation (CISH) and FISH in the determination of HER2 status in breast cancer," British journal of cancer, vol. 88, no. 10, pp. 1587-1591, 2003.
[59] A. S. Azevedo, C. Almeida, B. Pereira, P. Madureira, J. Wengel, and N. F. Azevedo, "Detection and discrimination of biofilm populations using locked nucleic acid/2′-O-methyl-RNA fluorescence in situ hybridization (LNA/2′ OMe-FISH)," Biochemical engineering journal, vol. 104, pp. 64-73, 2015.
[60] S. Fontenete et al., "Application of locked nucleic acid-based probes in fluorescence in situ hybridization," Applied Microbiology and Biotechnology, vol. 100, no. 13, pp. 5897-5906, Jul 2016.
[61] F. Silvia, B. Joana, M. Pedro, F. Ceu, W. Jesper, and A. N. Filipe, "Mismatch discrimination in fluorescent in situ hybridization using different types of nucleic acids," Applied Microbiology and Biotechnology, vol. 99, no. 9, pp. 3961-3969, May 2015.
[62] F. Wang et al., "RNAscope A Novel in Situ RNA Analysis Platform for Formalin-Fixed, Paraffin-Embedded Tissues," Journal of Molecular Diagnostics, vol. 14, no. 1, pp. 22-29, Jan 2012.
[63] E. Jensen, "Technical review: In situ hybridization," The Anatomical Record, vol. 297, no. 8, pp. 1349-1353, 2014.
[64] J. Ge, L. L. Zhang, S. J. Liu, R. Q. Yu, and X. Chu, "A Highly Sensitive Target-Primed Rolling Circle Amplification (TPRCA) Method for Fluorescent in Situ Hybridization Detection of MicroRNA in Tumor Cells," Analytical Chemistry, vol. 86, no. 3, pp. 1808-1815, Feb 2014.
[65] R. M. Lequin, "Enzyme immunoassay (EIA)/enzyme-linked immunosorbent assay (ELISA)," Clinical chemistry, vol. 51, no. 12, pp. 2415-2418, 2005.
[66] J. E. Butler, "Enzyme-linked immunosorbent assay," Journal of immunoassay, vol. 21, no. 2-3, pp. 165-209, 2000.
[67] K. Yue, T. N. Trung, Y. Zhu, R. Kaldenhoff, and L. Kai, "Co-translational insertion of aquaporins into liposome for functional analysis via an E. coli based cell-free protein synthesis system," Cells, vol. 8, no. 11, p. 1325, 2019.
[68] M. Z. Levine, N. E. Gregorio, M. C. Jewett, K. R. Watts, and J. P. Oza, "Escherichia coli-based cell-free protein synthesis: protocols for a robust, flexible, and accessible platform technology," JoVE (Journal of Visualized Experiments), no. 144, p. e58882, 2019.
[69] P. C. Zamecnik, I. D. Frantz Jr, R. B. Loftfield, and M. L. Stephenson, "Incorporation in vitro of radioactive carbon from carboxyl-labeled dl-alanine and glycine into proteins of normal and malignant rat livers," Journal of Biological Chemistry, vol. 175, no. 1, pp. 299-314, 1948.
[70] E. F. Gale and J. P. Folkes, "Effect of nucleic acids on protein synthesis and amino-acid incorporation in disrupted staphylococcal cells," Nature, vol. 173, no. 4417, pp. 1223-1227, 1954.
[71] A. Bank and P. Marks, "Protein synthesis in a cell free human reticulocyte system: ribosome function in thalassemia," The Journal of clinical investigation, vol. 45, no. 3, pp. 330-336, 1966.
[72] A. Marcus and J. Feeley, "Ribosome activation and polysome formation in vitro: requirement for ATP," Proceedings of the National Academy of Sciences of the United States of America, vol. 56, no. 6, p. 1770, 1966.
[73] S. Chong, "Overview of cell‐free protein synthesis: historic landmarks, commercial systems, and expanding applications," Current protocols in molecular biology, vol. 108, no. 1, pp. 16.30. 1-16.30. 11, 2014.
[74] E. D. Carlson, R. Gan, C. E. Hodgman, and M. C. Jewett, "Cell-free protein synthesis: applications come of age," Biotechnology advances, vol. 30, no. 5, pp. 1185-1194, 2012.
[75] L. S. Cohen and G. P. Studzinski, "Correlation between cell enlargement and nucleic acid and protein content of HeLa cells in unbalanced growth produced by inhibitors of DNA synthesis," Journal of Cellular Physiology, vol. 69, no. 3, pp. 331-339, 1967.
[76] L. P. Lim et al., "The microRNAs of Caenorhabditis elegans," Genes & Development, vol. 17, no. 8, pp. 991-1008, 2003.
[77] G. Yi, V. P. Brendel, C. Shu, P. Li, S. Palanathan, and C. Cheng Kao, "Single nucleotide polymorphisms of human STING can affect innate immune response to cyclic dinucleotides," PlOS ONE, vol. 8, no. 10, p. e77846, 2013.
[78] W. Chen, N. Ma, T. Li, L. Su, and Y. Chang, "MicroRNA in situ hybridization with phosphate-methylated oligonucleotides (nDNA) probe," New Biotechnology, vol. 44, p. S147, 2018.
[79] A. Malgoyre, S. Banzet, C. Mouret, A. X. Bigard, and A. Peinnequin, "Quantification of low-expressed mRNA using 5′ LNA-containing real-time PCR primers," Biochemical and biophysical research communications, vol. 354, no. 1, pp. 246-252, 2007.
[80] M. O. Urbanek, A. U. Nawrocka, and W. J. Krzyzosiak, "Small RNA detection by in situ hybridization methods," International journal of molecular sciences, vol. 16, no. 6, pp. 13259-13286, 2015.
[81] J. F. Lima et al., "Targeting miR-9 in gastric cancer cells using locked nucleic acid oligonucleotides," Bmc Molecular Biology, vol. 19, Jun 2018, Art. no. 6.
[82] X.-F. Le, O. Merchant, R. C. Bast, and G. A. Calin, "The roles of microRNAs in the cancer invasion-metastasis cascade," Cancer Microenvironment, vol. 3, no. 1, pp. 137-147, 2010.

指導教授

陳文逸(Wen-Yih Chen)

審核日期

2022-7-28

推文