April Binder (Biological Sciences)
The ability to isolate quality RNA from a sample is an integral component for studying gene expression. By studying transcribed RNA, researchers can observe changes in gene expression and investigate the role of specific genes that are expressed in different cells in order to understand cell specialization and function. RNA isolation involves isolating RNA from biological samples and is a prerequisite procedure for further assays including cDNA synthesis and real-time PCR (RT-qPCR). Isolated RNA can also be quantified using fluorescence and UV based methods, such as a microplate reader. In this procedure, TRIzol reagent was used to isolate RNA from white and brown adipose tissue collected from mice in order to perform qualitative and quantitative RNA analysis, cDNA synthesis, and RT-qPCR. TRIzol is a phenol based reagent generally used to isolate RNA from cell and tissue samples. TRIzol contains phenol and guanidinium isothiocyanate that solubilize cell materials before the addition of chloroform, which causes phase separation into an aqueous RNA phase, a DNA interface, and a protein organic phase. The aqueous phase is then separated and precipitated into a pellet using isopropanol and washed in diethyl pyrocarbonate (DEPC) water before being left to dry. Finally, the RNA pellet is resuspended in DEPC water to be used in further assays. Isolating RNA from adipose tissue can be challenging compared to other tissue types and cells. This presentation demonstrates the protocol that was determined to successfully isolate high quality RNA from adipose tissues and then confirm primers to be used for qRT-PCR.
Keywords: RNA isolation, molecular techniques