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Ribonucleic Acid (RNA) is essential for genetic regulation and expression. That’s why its extraction is a major part of gene expression analyses. In many neuroscience studies, animal models such as mice are used. Accordingly, you may find that you must extract RNA from mouse brain tissue at some point or other. While the extraction procedure itself isn’t complicated, two challenges arise when trying to extract RNA from mouse brain samples. The first challenge is that working with RNA is tougher than working with DNA because RNA is inherently less stable due to its chemical structure. It’s unstable at higher temperatures (> 65°C) and can be degraded in the presence of metal ions or in alkaline conditions. Additionally, RNases are more ubiquitous in standard laboratory conditions than DNases. Even the slightest exposure to RNase can impact RNA stability. RNases are problematic because they are resistant to high temperatures and chemical treatment. The second challenge faced when extracting RNA from mouse brain is the nature of the tissue. In general, isolating intact total RNA is more difficult when processing certain problematic tissues, such as lipid and protein-rich tissues and fibrous tissues. The brain falls under this category due to its high lipid content. This guide outlines the main precautions you should take to overcome these challenges. It’s meant to supplement your main extraction lab manual. If you would like to read further on the topic, please check references 3 and 4.
A pellet will form at the bottom of the tube containing extracellular material, high molecular weight DNA, and polysaccharides, while a layer of fat collects above the supernatant. Discard the fatty layer and collect the supernatant to continue extraction according to the directions of your extraction kit. 5. Storage:
Ngs Lib Prep Treat with DNase to remove residual genomic DNA.