Structural Biochemistry/Nucleic Acid/RNA/RNA Extraction

RNA Extraction

RNA extraction is a technique of isolating and purifying RNA from in-vivo tissues and samples. There are several ways of extracting RNA. The presence of ribonucleases enzymes within the tissue cells complicates the extraction and purification process by quickly degrading the isolated RNA. Isolated and purified RNA can be used to detect gene expression, biomarkers, drug efficacy, and much more.

Trizol RNA extraction

File:RNA extraction.jpg First, homogenize the sample tissue in Trizol solution using a vortex mixer. The speed of mixing is very important because the tissue can only be homogenized when mixing at high speed, however, friction may cause heat, which may accelerate RNA degradation. Add chloroform to the finely grinded mixture to perform a liquid phase separation. For every milliliter of Trizol reagent, add 0.2 mL of chloroform. Cap and shake the mixtures vigorous for 15 seconds and incubate them at room temperature. Centrifuge the mixtures at no more than 14,000 rpm for 15 minutes at 2-80C. After centrifugation, the mixture separates into three distinct layers, the lower red chloroform layer, an interphase layer of remaining tissues and fat, and a clear upper aqueous layer. RNA remains in the aqueous layer exclusively. Transfer the aqueous layer to another container. Wash and precipitate the RNA by using isopropyl alcohol. Add 0.5mL of isopropyl alcohol to every mL of Trizol reagent. Incubate at room temperature for 10 minutes and centrifuge again for 4 minutes in RNA elution plates. Remove all supernatant and wash the RNA again with ethanol. Wash the RNA subsequently with buffers. Make sure to dry off any remaining alcohol because they lower the quality of RNA and promote RNA degradation. The last wash should contain RNase free water to elude out the isolated and purified RNA.

Reference

1. Gottshall, Susan L., Saban Tekin, and Peter J. Hansen. "EXTRACTION AND PURIFICATION OF TOTAL RNA USING TRIzol OR TRI REAGENT." (n.d.): n. pag. Web. 15 Nov. 2012. <http://www.animal.ufl.edu/hansen/protocols/RNA_extraction.htm>.