| Abstract: | 傳遞核醣核酸 ( transfer RNA , tRNA ) 於蛋白質生成上扮演重要的角色，被稱之為第二遺傳密碼，近來研究發現癌症疾病之發展與tRNA上所攜帶的特定胺基酸有密切的關係，可得知四種 tRNA 分別與不同癌細胞相關:Arg-tRNA與尿路上皮癌細胞，Tyr-tRNA 與卵巢癌細胞，Met-tRNA、Phe-tRNA 與乳癌。本實驗方法為在電滲流存在下以毛細管電泳暨雷射誘發螢光分離攜帶不同胺基酸的 tRNA 3’ 端處，此方法利用分子量為 8,000,000 Da 之聚環氧乙烷 ( PEO ) 作為分離 tRNA 的篩分介質，並將尿素溶解在聚合物溶液中，使特定長度的短鏈雙股寡核苷酸變性，將雙股的位置解開成單股。本實驗以 Tyr-tRNA、Phe-tRNA、Met-tRNA 及 Arg-tRNA 做為分析物並設計由四種長度不同的橋樑 DNA ( bridge DNA ) 與總核醣核酸 ( total RNA ) 中的tRNA 先進行雜合及 Ligation 後再以電泳分離。此方法可用於檢測人體中氨醯 tRNA( aminoacyl-tRNA ) 的含量，進而推測與癌症的相關性。先前文獻之偵測方法多為使用微陣列、反轉錄聚合酶連鎖反應與北方墨點法，但前兩種方法所需之成本相對較高，而北方墨點法的靈敏度較低，因此本實驗發展出一種成本較低且樣品不需經由PCR放大，便可直接對樣品進行偵測的方法。
Transfer RNA (tRNA) plays an important role in the production of protein, several studies have found that the development of cancer diseases has the closely connection with specific amino acid carried on tRNA, here are four tRNAs are associated with different cancer cells: Arg-tRNA for urothelial carcinoma, Tyr-tRNA for ovarian cancer and Met-tRNA, Phe-tRNA for breast cancer. This experiment is based on the capillary electrophoresis with laser-induced fluorescence in the presence of electroosmotic flow to separate the different amino acid to cancer on the 3’-end of tRNA. The method utilizes the poly ethylene oxide (Mave 8,000,000) as the sieving media to separate the four tRNAs. To denature the specific length oligonucleotide, we use urea dissolved in the polymer solution. In this experiment, Tyr-tRNA, Phe-tRNA, Met-tRNA, and Arg-tRNA are taken as analytes, and we design four different length of bridge DNA to do hybridization and ligation with total RNA then we can separate by electrophoresis. This approach can be applied on testing the amount of aminoacyl-tRNA in human body, and then predict the relation with the cancer. According to the detection method of the previous articles mostly use Microarray, RT-PCR and Northern Blot; however, the cost of the first two methods is high, while the sensitivity of the Northern Blot is relatively low. Therefore, this experiment has developed a method that can directly detect the sample with low cost and doesn’t need to be amplified by PCR. |
