An RNase inhibitor is a protein that prevents the activity of enzymes known as ribonucleases (RNases). These inhibitors are crucial tools in molecular biology, protecting RNA molecules from degradation. Their primary purpose is to maintain RNA sample integrity, which is important for various laboratory procedures and ensures accurate experimental results.
The Role of RNase Inhibitors in RNA Protection
RNA molecules are inherently unstable and highly susceptible to degradation by RNases, which are ubiquitous enzymes. These enzymes break down RNA into smaller components, often as part of natural cellular processes like RNA processing and gene regulation. However, RNases are also commonly present as contaminants in laboratory settings, originating from sources like human skin, dust, or improperly cleaned equipment.
RNase inhibitors counteract this degradation by binding tightly to RNases, effectively neutralizing their enzymatic activity. Most commercial RNase inhibitors are recombinant proteins that form stable, non-covalent complexes with RNases. This binding prevents the RNases from accessing and cleaving RNA molecules, thereby preserving the RNA’s structure and function for downstream applications, enabling progress in fields such as gene expression studies and the development of RNA-based therapies.
Determining the Optimal RNase Inhibitor Concentration
Determining the precise amount of RNase inhibitor to use is a common query, and the “optimal” concentration often varies based on the specific laboratory application and the expected level of RNase contamination. RNase inhibitors are typically measured in units (U), where one unit is generally defined as the amount required to inhibit a certain quantity of RNase A, a common and potent RNase. For many common molecular biology applications, such as RNA extraction, cDNA synthesis, reverse transcription-polymerase chain reaction (RT-PCR), and RNA storage, typical recommended concentrations range from 1 to 2 units per microliter (U/µl) in the final reaction volume.
However, the exact amount can depend on factors like the specific protocol being followed, the volume of the reaction, the purity of other reagents, and the anticipated RNase contamination within the sample. It is always advisable to consult the manufacturer’s recommendations for the specific RNase inhibitor product being used, as concentrations and unit definitions can differ between brands like Promega’s RNasin, Invitrogen’s SUPERase-In, or New England Biolabs’ Murine RNase Inhibitor.
Adding too little inhibitor may result in incomplete protection and RNA degradation, while adding an excess amount is generally not harmful to the reaction components but can be wasteful. The goal is to provide sufficient inhibition to overcome potential RNase activity without unnecessary overuse.
Practical Application and Handling
For effective RNA protection, the RNase inhibitor should be introduced into the sample as early as possible in the experimental workflow. Ideally, it should be added immediately after cell lysis or sample collection, preventing any initial RNA degradation. This ensures quick neutralization of any RNases released or introduced during initial handling.
Proper handling and storage of RNase inhibitors are also important to maintain their activity. These proteins are often sensitive to various conditions, including temperature fluctuations. Most RNase inhibitors are typically stored at -20°C to preserve their efficacy. Repeated freeze-thaw cycles should be avoided, as this can lead to a loss of activity over time. Additionally, maintaining an overall RNase-free environment during all steps of RNA handling, including using sterile, RNase-free consumables and reagents, complements the inhibitor’s action and further safeguards RNA integrity.
Troubleshooting RNA Degradation
Despite the use of RNase inhibitors, RNA degradation can sometimes occur, indicated by signs such as smearing rather than distinct bands on a gel electrophoresis image. When degradation persists, it often points to issues beyond merely insufficient RNase inhibitor concentration.
Potential reasons for continued RNA degradation include extremely high levels of RNase contamination in the sample or environment, which may overwhelm the inhibitor. Improper sample handling, such as prolonged exposure to room temperature or inadequate storage, can also contribute to degradation. Problems with other reagents, like using water or buffers that are not truly RNase-free, can introduce active enzymes. In such cases, re-evaluating the entire RNA isolation protocol, ensuring all reagents are certified RNase-free, and considering the use of a fresh or higher quality inhibitor stock are important troubleshooting steps.