Analysis of the total RNA integrity by capillary electrophoresis with laser-induced fluorescence in the presence of electroosmotic flow and assembly of dark field microscope to detect gold nanoparticles

• Main Authors: Ya-Chu Shih, 施雅筑
• Other Authors: Po-Ling Chang
• Format: Others
• Language: zh-TW
• Published: 2013
• Online Access: http://ndltd.ncl.edu.tw/handle/62197309421855767881

碩士 === 東海大學 === 化學系 === 101 === RNA integrity plays an important role in RNA studies because poor RNA quality may have a great impact on downstream methodologies. This study proposed a rapid, and cost-effective method to analyze RNA integrity based on capillary electrophoresis with laser-induced fluorescence(CE-LIF)in the presence of electroosmotic flow. This proposed method used the poly(ethylene) oxide (Mave 4,000,000 Da) as sieving matrix for total RNA separation. The ethidium bromide (μg/mL) was dissolved in polymer solution as interchelating dye for the on column fluorescent labeling. The separation of the 28S rRNA, 18S rRNA, 5.8S rRNA, 5S rRNAs and tRNA from cell extracted human total RNA was accomplished within 25 minutes. In this work, online concentration technique was used to increase the sensitivity by larger volume sample injection. The lower detectable concentration of 28S and 18S rRNAs is 100 pg/μL. In addition, the temperature-induced degradation of total RNAs could be monitored by CE-LIF. The result demonstrated the separation of total RNA by CE-LIF in the presence of EOF is useful for determination of RNA quality. The second part of this thesis is to build a dark field microscope to detect gold nanoparticles. By using high numerical objective, appropriately field stop and iris, observation of brownian motion of 32 nm gold nanoparticles was successfully recorded by commercial CCD camera. This result indicated the proposed instrument may potentially be use for sensitive detection of nanoparticle-based nanosensing.