RNA is isolated by separating it from other cellular components like proteins and DNA, often using a combination of chemical and physical methods. A common approach involves using guanidinium thiocyanate (GuSCN), phenol, and alcohol precipitation.
Here's a breakdown of the process:
-
Cell Lysis and Denaturation: The process starts with lysing the cells or tissue to release the RNA. Guanidinium thiocyanate (GuSCN) is often used as a strong chaotropic agent. GuSCN denatures proteins, including RNases (enzymes that degrade RNA), ensuring the integrity of the RNA.
-
Phase Separation (Phenol-Chloroform Extraction):
- Following cell lysis, phenol is added. The pH is critical; maintaining a pH around 7.0 +/- 0.2 favors RNA partitioning into the aqueous phase.
- Phenol and chloroform are added to the lysate. This mixture is then centrifuged, resulting in two distinct phases: an aqueous phase (containing the RNA) and an organic phase (containing DNA and proteins).
- The RNA, being more soluble in water, remains in the upper aqueous phase. The DNA and proteins, denatured by the phenol, partition into the lower organic phase.
-
RNA Precipitation:
- The aqueous phase containing the RNA is carefully removed.
- RNA is precipitated from the aqueous phase by adding a salt (e.g., sodium acetate) and an alcohol (either ethanol or isopropanol). The salt neutralizes the negative charge of the RNA, and the alcohol reduces the polarity of the solution, causing the RNA to aggregate and precipitate.
- The mixture is then incubated at a low temperature (e.g., -20°C or -80°C) to enhance precipitation.
- The precipitated RNA is collected by centrifugation.
-
Washing:
- The RNA pellet is washed with ethanol (e.g., 70% ethanol) to remove residual salts and other impurities.
-
Resuspension:
- Finally, the RNA pellet is air-dried briefly and then resuspended in a suitable buffer, such as RNase-free water or TE buffer, ready for downstream applications.
Alternative Methods:
While phenol-chloroform extraction is widely used, other methods for RNA isolation exist, including:
- Column-based purification: These methods use silica-based or other matrices to bind RNA selectively under specific conditions. After washing away impurities, the RNA is eluted from the column. These methods are often faster and more convenient than phenol-chloroform extraction, although they may sometimes yield lower amounts of RNA.
- Direct lysis methods: Some kits contain reagents that lyse cells and allow for direct RNA binding to a purification column, streamlining the process.
- Chloroform alone: While less common, using chloroform alone (without phenol) has been suggested for bacterial RNA extraction.
In summary, RNA isolation typically involves cell lysis, separation of RNA from other biomolecules using methods like phenol-chloroform extraction or column purification, precipitation of RNA, washing, and resuspension in a suitable buffer.
