Developing An Isolation Method For Small Rns From Plasma Samples

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.