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研究生:林配奾
研究生(外文):Pei-Hsien Lin
論文名稱:血液存放溫度與時間對於血液中去氧核醣核酸與核醣核酸的影響
論文名稱(外文):The effects of storage temperature and duration of blood samples on DNA and RNA qualities
指導教授:吳長益李松洲
指導教授(外文):Chang-Yi WuSung-Chou Li
學位類別:碩士
校院名稱:國立中山大學
系所名稱:生物科學系研究所
學門:生命科學學門
學類:生物學類
論文種類:學術論文
論文出版年:2018
畢業學年度:106
語文別:中文
論文頁數:44
中文關鍵詞:儲存時間去氧核醣核酸甲基化去氧核醣核酸品質核醣核酸品質RIN 值儲存溫度即時定量聚合酶連鎖反應
外文關鍵詞:storage temperatureDNA qualityRIN valueDNA methylationstorage durationqPCRRNA quality
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去氧核醣核酸及核醣核酸的品質對於生物醫學的研究是至關重要的,例如:微陣列基因檢測或次世代定序。高品質的去氧核醣核酸及核醣核酸樣本可以確保這些測試或研究的正確性。在此我們研究儲存血液的溫度和時間對於白血球的去氧核醣核酸及核醣核酸品質的影響。首先將血液檢體儲存在不同溫度及不同時間,之後利用微流體電泳分析儀檢測核醣核酸的完整度數值 (RNA Integrity Number, RIN) 和即時聚合酶鏈式反應進行核醣核酸的品質檢測,或是利用微流體電泳分析儀、點墨跡實驗和即時焦磷酸序列分析儀觀察去氧核醣核酸的穩定性。實驗結果顯示血液儲存時間超過 24 小時會導致核醣核酸的品質明顯下降。將血液儲存在低溫下 (4℃) 並不能防止核醣核酸的降解。若是將血液樣品超低溫冷凍儲存 (-80℃) 會顯著的損害核醣核酸的品質。對於去氧核醣核酸而言,在 15 天的觀察當中不同溫度的儲存條件並不會導致品質的下降。然而透過即時焦磷酸序列分析儀觀察到C-C motif chemokine 22 (CCL22) 基因上的甲基化程度在存放時間超過三天後有顯著的改變。利用點墨跡實驗發現整體的去氧核醣核酸的甲基化程度發生改變。綜合上述結果,24 小時內的儲存時間對於核醣核酸的品質足以通過測定次世代定序的要求 (RIN≧8)。若是基於微陣列基因檢測之樣品要求 (RIN≧7) 則可接受樣品儲存時間在 32 小時內。此外,儘管去氧核醣核酸的品質相對穩定,但是總萃取量會有明顯的減少,其甲基化程度的改變也值得關注。
DNA and RNA quality are critical for biomedical studies such as microarray and NGS. Since high-quality DNA and RNA samples guarantee the correctness of these tests and/or studies, we investigated the effects of storage temperature and storage duration of DNA and RNA qualities from blood. Blood samples were stored for different durations and at different temperatures, followed by the examinations on RNA quality, qPCR, DNA quality and DNA methylation. For RNA, we observed obvious quality decline with storage duration longer than 24 hours base on bioanalyzer and qPCR results. Storage at low temperature does not keep RNA samples from degradation. Storage of whole blood samples in freezer dramatically damage RNA. For DNA, quality decline was not observed even with storage duration for 15 days. However, DNA methylation status significantly altered with storage duration longer than three days through dot blot and pyromark. In summary, storage duration within 24 hours is critical for collecting high-quality RNA samples for next-generation sequencing (NGS) assays (RIN≧8). If microarray assays are expected (RIN≧7), storage duration within 32 hours is acceptable. Although DNA is resistant within 15 days when kept in whole blood, DNA quantity dramatically decreases owing to white blood cells (WBCs) lysis. In addition, duration for more than three days significantly alter DNA methylation status, globally and locally. Our result provides a reference for dealing with blood samples.
論文審定書 i
誌謝 ii
中文摘要 iii
Abstract iv
目錄 v
圖次 vi
表次 vii
第一章緒論 1
1.1 臨床檢體於研究之應用 1
1.2 影響檢體品質的因素 1
第二章實驗材料與方法 3
2.1 檢體採集與製備 3
2.2 核酸萃取 3
2.3 超微量分光光度計 (NanoDrop 2000, Thermo Fisher Scientific) 4
2.4 微流體電泳分析儀 (Agilent 2100 Bioanalyzer, Agilent, CA, USA) 4
2.5 微流體電泳分析儀 (Agilent 4200 TapeStation, Agilent, CA, USA) 6
2.6 反轉錄聚合酶連鎖反應 (reverse transcription-PCR, RT-PCR) 7
2.7 即時定量聚合酶連鎖反應 (Quantitative real time polymerase chain reaction, qPCR) 8
2.8 即時焦磷酸序列分析儀檢測甲基化程度 9
2.9 點墨跡實驗 (Dot blot hybridization) 11
第三章實驗結果 12
3.1 血液保存時間對於核糖核酸品質之影響 12
3.2 血液保存時間對於去氧核糖核酸品質之影響 13
3.3 血液保存時間對於甲基化之影響 15
第四章討論 17
參考文獻 20
附錄一 22
附錄二 36
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