RNA hydrolysis, the degradation of RNA molecules, can be prevented by controlling several key factors. The most crucial aspect is maintaining a slightly acidic to neutral pH environment.
Here's a breakdown of methods to prevent RNA hydrolysis:
-
Maintain Optimal pH:
- Avoid Alkaline Conditions: RNA is particularly susceptible to hydrolysis in alkaline solutions. Therefore, avoid storing or working with RNA at pH levels higher than 7.5.
- Use Appropriate Buffers: Utilize buffers such as citrate buffer (pH 6) or Tris-EDTA (TE) buffer (pH 7.5) for RNA storage and manipulation. These buffers help maintain a stable pH and prevent degradation.
-
Control Temperature:
- Store at Low Temperatures: Store RNA at -20°C or -80°C to significantly slow down hydrolysis.
- Avoid Repeated Freeze-Thaw Cycles: Minimize freeze-thaw cycles as they can contribute to RNA degradation. Aliquot RNA into smaller volumes to avoid thawing the entire stock each time.
-
Minimize RNase Contamination:
- Use RNase-Free Reagents and Consumables: Employ reagents, water, tubes, and pipette tips that are certified RNase-free.
- Wear Gloves: RNases are present on human skin, so always wear gloves when handling RNA. Change gloves frequently.
- Clean Work Surfaces: Regularly clean work surfaces with RNase-decontaminating solutions.
-
Use Additives (Optional):
- RNase Inhibitors: Consider adding RNase inhibitors to your RNA solutions. These inhibitors specifically block the activity of RNases, preventing them from degrading the RNA.
- Chelating Agents: EDTA, commonly found in TE buffer, chelates divalent cations such as Mg2+, which are required for RNase activity.
In summary, preventing RNA hydrolysis involves controlling pH, temperature, and RNase contamination. Storing RNA in slightly acidic to neutral buffers at low temperatures while using RNase-free practices is crucial for maintaining RNA integrity.
