Cleaning or purifying RNA is essential for many molecular biology applications. The primary goal is to remove contaminants like proteins, salts, and other cellular components, leaving you with high-quality RNA. Here’s how it's commonly done:
RNA Purification Methods
There are two main methods for precipitating and purifying RNA. Both are effective, but alcohol precipitation is the most widely used.
1. Alcohol Precipitation
Alcohol precipitation is a popular method for concentrating and purifying RNA. It works by reducing the solubility of RNA in aqueous solution, causing it to precipitate out. This process involves these key steps:
- Add Salt: Initially, a salt solution (like sodium acetate or ammonium acetate) is added to neutralize the negative charges of the RNA's phosphate backbone. This neutralizes the charges which allow RNA to become less soluble.
- Add Alcohol: Then, an alcohol, typically ethanol or isopropanol is added. The alcohol reduces the RNA's solubility in the aqueous solution. This causes the RNA molecules to clump together.
- Incubation: The mixture is incubated at a low temperature. The cold temperature makes it easier for RNA to come out of solution.
- Centrifugation: After incubation, the sample is centrifuged. The precipitated RNA forms a pellet at the bottom of the tube.
- Washing: The alcohol and any remaining contaminants are removed by washing the pellet with a suitable buffer like 70% ethanol.
- Drying: The RNA pellet is air-dried to remove any residual alcohol.
- Resuspension: Finally the RNA is resuspended in an appropriate buffer like RNase-free water or TE buffer.
2. Lithium Chloride Precipitation
Another method for precipitating RNA involves using lithium chloride (LiCl). It's less common, but can be effective. This method works on the principle that LiCl has a selective ability to precipitate RNA.
- LiCl Addition: A high concentration of LiCl solution is added to the RNA sample.
- Incubation: The mixture is incubated, often at low temperatures, to allow the RNA to precipitate out.
- Centrifugation: Similar to alcohol precipitation, the sample is centrifuged, and the RNA forms a pellet.
- Washing: The pellet is washed with cold LiCl to remove impurities.
- Resuspension: Finally the RNA is resuspended in an appropriate buffer like RNase-free water or TE buffer.
Summary of RNA Cleaning Methods
| Method | Principle | Common Use |
|---|---|---|
| Alcohol Precipitation | Reducing RNA solubility with alcohol and salt; commonly used to precipitate RNA. | Widely used, effective for concentrating and purifying RNA. |
| Lithium Chloride (LiCl) Precipitation | Selective RNA precipitation with LiCl | Less common, but still used for specific purification or when alcohol is not desirable. |
Considerations for RNA Purification
- RNase-Free Environment: It's crucial to work in an RNase-free environment to prevent RNA degradation. Use RNase-free materials.
- Cold Temperatures: Maintaining cold temperatures throughout the purification process is very important to prevent RNA degradation.
- Choosing the Right Method: The appropriate method will depend on the starting material, amount of RNA, and downstream application. Alcohol precipitation is more commonly used.
By following these methods carefully, you can effectively clean and purify RNA for your research or experiments.
