| dc.description.abstract | Recently, the utility of circulating micro RNAs (miRNAs) has proposed a
dominant method to pre-diagnose and treat diseases. Although a variety of
commercial kits for RNA extraction are now available, they have a limit at
collecting total RNAs. Some suggested a Trizol-based method specific to small
RNAs, which applied lithium chloride (LiCl) in the precipitation step. However,
other studies proposed that LiCl cannot effectively precipitate small RNAs in
contrast to magnesium chloride (MgCl2). This project aims to modify and develop
a more efficient extraction method for circulating small RNAs, including miRNAs.
After choosing the most suitable MgCl2 concentration; the comparison between
using MgCl2, LiCl, and isolation kit (PureLink RNA Mini Kit) was conducted. The
modified method was then validated through testing RNA size range, the
feasibility for Reverse Transcription quantitative Polymerase Chain Reaction (RTqPCR), and the stability. The result revealed that the final precipitation using the
mixture of 0.5M MgCl2 and ethanol provided the highest yield, five-time and tentime higher than LiCl and kit respectively. Subsequently, no band detected in
automated electrophoresis indicating minimal large RNA contamination, and high
efficiency of the modified method in RT-qPCR was also confirmed with 100%
successful in amplifying miR-16 with Cq of 23.64±2.02. Thereby, this method is
promised to be an effective way to extract circulating small RNAs with high
quality and quantity for downstream quantification analysis. | en_US |
