How to Purify RNA?

Purifying RNA involves several critical steps designed to isolate RNA from a complex biological sample while preventing degradation. The primary goal is to obtain high-quality RNA, free from contaminants like proteins, DNA, and other cellular components. Here's a breakdown of the common approaches and considerations:

Core Principles of RNA Purification

The core principle of RNA purification is to separate RNA molecules from other cellular components while ensuring the RNA remains intact. Most traditional methods incorporate RNase inhibitory agents, which helps prevent RNA degradation. These agents typically include strong denaturants like guanidine salts, sodium dodecylsulfate (SDS), or phenol-based compounds.

Common RNA Purification Methods

1. Organic Extraction (e.g., TRIzol Method)

This method employs a mixture of phenol and guanidine thiocyanate to lyse cells and denature proteins. The procedure follows these basic steps:

• Cell Lysis: The biological sample is homogenized in a solution containing guanidine thiocyanate, which disrupts cell membranes and inactivates RNases.

• Phase Separation: Chloroform is added, and the mixture is centrifuged to separate the aqueous phase (containing RNA) from the organic phase (containing proteins and lipids) and the interphase.

• RNA Precipitation: The aqueous phase is collected, and RNA is precipitated with isopropanol.

• Washing: The precipitated RNA is washed with ethanol to remove residual salts.

• Resuspension: The RNA pellet is dissolved in an appropriate buffer.

  Advantages: Effective for various sample types, relatively cost-effective.
  Disadvantages: Use of toxic chemicals, time-consuming, may require multiple steps.

2. Silica-Based Column Purification

This method uses specialized columns containing silica membranes to bind RNA selectively, with the following basic steps:

• Cell Lysis and RNA Binding: Cells are lysed using a buffer that also allows RNA to bind to the silica membrane within the column.

• Washing: Various washing solutions are used to remove proteins, DNA, and other contaminants, while RNA remains bound.

• Elution: The purified RNA is eluted from the column using a low-salt buffer or water.

  Advantages: Fast, convenient, no need for toxic chemicals.
  Disadvantages: Can be more expensive than organic extraction, some RNA loss may occur if proper handling is not ensured.

3. Magnetic Bead-Based Purification

In this method, magnetic beads functionalized to bind RNA are used. The steps include:

• Cell Lysis and RNA Binding: Similar to column-based methods, the cells are lysed and the RNA binds to the magnetic beads.

• Magnetic Separation and Washing: The beads are separated from the lysate using a magnet, allowing washing steps to remove contaminants.

• Elution: The purified RNA is eluted from the beads using a buffer.

  Advantages: Adaptable for automation, scalable, efficient for small sample volumes.
  Disadvantages: Can be more expensive than organic extraction, some loss of RNA may occur due to the binding process.

Considerations for Successful RNA Purification

• RNase Prevention: Always work in an RNase-free environment, using sterile, RNase-free consumables. Wear gloves and use RNase inhibitor solutions where necessary. As mentioned previously, most RNA purification protocols include RNase inhibitory agents.
• Sample Preparation: Proper handling and quick processing of samples minimize RNA degradation.
• Choice of Method: The best method depends on the type and size of the sample, the required yield, and budget constraints.
• RNA Integrity: Evaluate RNA quality using methods like gel electrophoresis or microfluidics to ensure the integrity of the purified product.

Summary Table

By following these methods and keeping the considerations in mind, you can successfully purify RNA for downstream applications.