| dc.description.abstract | MicroRNAs (miRNAs) are promising biomarkers that could be applied on the diagnosis and treatment of different diseases. For the miRNA diagnosis assays, the purity and quality of miRNA samples are important in pre-analytical steps. In this study, we modified the commercial RNA isolation kits for miRNA extraction from the culture cell to improve the purity and amount of miRNA. First of all, we investigated the binding mechanism of nucleic acid and silica membrane of silica spin column. Consequently, we found that nucleic acid are much easier binding with silica surface at low pH value, presence of chaotropic salt and alcohol solution condition. Moreover, there are several methodologies could increase the binding affinity between RNA and silica surface. In these methodologies, adjusting the solution polarity, and adding divalent cation in binding process can promote RNA adsorption on silica surface. For desorption, we raised the elution temperature to break the bonding and raise the pH value to enhance the repulsive force between RNA and silica membrane to increase the RNA recovery. By using the real-time polymerase chain reaction (RT-qPCR) and nanowire field effect transistor (NWFET), we could compare the relative ef?ciencies of modified protocol and the original one. Using spike-in synthesis exogenous miRNA, mir-39, evaluate the recovery of modified process.
The result show that using the TE buffer for the elution buffer and increasing the elute temperature will increasing the miRNA isolation efficiency. Moreover, the presence of the Ca2+ cation did not improve the miRNA isolation process. The ethanol concentration 65% (v/v) for miRNA isolation have the better efficiency than the 60% (v/v) and 70% (v/v). In the end, we combine three results as the new protocol, the new protocol isolation efficiency is 2 fold better than the original protocol. The NWFET was applied to detect the miRNA, the result was compared with the RT-qPCR for miRNA quantification. The result showed that the NWFET can distinguish different concentration of target miRNA in the cell extraction. As for the result, the NWFET is the potential tool for detecting the miRNA. | en_US |