RNase Testing
The RNase Assay, short for Ribonuclease Assay, is an analytical process in molecular biology. It involves the assessment of ribonuclease activity in biological samples and solutions. Ribonucleases (RNases) are enzymes that degrade ribonucleic acid (RNA) molecules that are naturally occurring and found across various organisms, particularly in bacteria and fungi.
History of RNase Testing
The history of the RNase Assay dates to its initial development as a spectrophotometric method. This method was based on the observation that ribonucleases cause a gradual shift in the ultraviolet absorption spectrum of RNA substrates towards shorter wavelengths [1]. Over time, several traditional methods for analyzing RNase activity emerged, including radioactive label-based assay (reference), RNA-Pyronine Y complex assay [2], gel zymography [3]. methylene blue-based assay [4], and agarose gel electrophoresis-based assay [5].
The agarose gel electrophoresis RNase assay technique was commonly used to separate RNA fragments following degradation by RNases prior to newer quality control methods. In this process, fragments are detected by staining the gel with the intercalating dye, ethidium bromide, followed by visualization/photography under ultraviolet light. Figure 1 demonstrates an in-house gel electrophoresis RNase test performed at Boston BioProducts prior to current techniques.
Figure 1: RNase Activity assay developed by Boston BioProducts separates known RNA size values via a kilobase (kb) ladder (leftmost row), to determine cleaved RNA strands that appear to be smeared due to different sized regions that have been spliced by RNase.
Importance of Performing RNase Assay in Buffers, Reagents, and Media
RNase Assay is critically important in molecular biology due to the ubiquity of RNases in nature, and can contaminate various resources, including water sources, containers, and equipment used for preparing reagents. These sources of contamination pose a risk to molecular biology applications due to their frequent usage and can potentially lead to erroneous results and data. Since RNases are specific for RNA degradation, it's essential to ensure that these essential components of experiments are free from RNase contamination.
Figure 2: The Mechanism of RNase, and its affects in mRNA degradation. mRNA degradation, essential for cellular processes, operates in a precise manner. First, RNA-binding proteins identify structural anomalies. RNase enzymes then act as molecular "scissors", cleaving the compromised segment, and therefore preventing translation into dysfunctional proteins. Subsequent cellular processes remove and degrade the resulting mRNA fragment, preserving integrity. Regulatory networks govern RNase cleavage, exemplifying the cellular commitment to precision in mRNA quality control. This process is also known as a post-transcriptional regulatory mechanism in a biological context.
RNase Testing at Boston BioProducts
Boston BioProducts employs an ultrasensitive High Throughput Fluorometric RNase Detection assay system for RNase testing. This assay utilizes fluorescence-quenched oligonucleotide probes. These probes fluoresce upon exposure to ribonuclease digestion. It can also be visualized by illumination with UV light. By using positive and negative controls, samples then can be examined against expected specifications.
Considerations and Limitations of RNase Testing |
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| Clean Work Area and Equipment | Prioritize the cleanliness of workspaces, pipettes, and incubators by using nuclease decontamination solutions. |
| Protective Attire | Wear laboratory coats and gloves to prevent particulate materials from contaminating samples. Change gloves after touching potentially contaminated surfaces. |
| RNase-Free Materials | Use tips, tubes, and equipment known to be RNase-free to avoid contamination. |
| Biological Hood Usage | Perform the assay in a biological safety cabinet or a low-traffic area away from air vents or open windows to minimize external contamination. |
| Adjust pH and Salt Concentration | Ensure that the pH and salt concentration of samples are adjusted to optimal conditions for RNase assay, as these factors can affect enzyme activity. |
| Sterilization | Autoclave or sterile filter buffer solutions immediately after preparation to prevent contamination. |
Inhibitors and Activators |
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| pH of the Buffer Solutions | RNase activity is optimal at neutral pH (i.e., 7 to 8). Hence, it is important that the pH of the samples is adjusted to neutral pH before subjecting them to RNase assay |
| Ionic Concentration in the Buffer Solutions | Solutions with high ionic strength are known to inhibit RNases. Therefore, it is necessary that the samples with high salt concentrations are diluted (up to 5-10 mM) appropriately before performing the assay |
| Presence of Divalent Cations in Buffer Solutions | The presence of Ba2+, Co2+, Pb2+, As3+, and Cu2+ cations inhibit RNase activity. |
| Presence of Chelators in Buffer Solutions | Presence of chelators such as EGTA and EDTA inhibit RNase activity. |
| Presence of Reducing Agents in Buffer Solutions | The presence of reducing agents such as β-mercaptoethanol, dithiothreitol (DTT), dithioerythritol (DTE), and reduced glutathione (GSH) inhibit the RNase activity. |
| Presence of Chaotropic Agents | Chaotropic agents such as guanidine thiocyanate, guanidine hydrochloride and urea denature the RNases and thus inhibit their activity. |
| Gel Loading Buffers or Colored Solutions | Colored solutions such as Protein loading Dye or DNA loading dye solution interfere with DNase assays, In addition to the dyes in theses loading solutions the presence of SDS, EDTA, or β-mercaptoethanol further inhibits RNase activity. |
| DEPC Treatment | While DEPC treatment can inactivate RNases in some solutions, avoid using it in buffers containing primary and secondary/tertiary amines, such as Tris and HEPES, as it may not be effective and could impact buffering capabilities. |
In addition to RNase Testing, Boston BioProducts provides a comprehensive set of QC tests for custom reagents. Learn more about custom reagent development services.
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References:
- Kunitz M. (1946). A spectrophotometric method for the measurement of ribonuclease activity. J Biol. Chem.;164(2):563–8.
- Lee, P.Y., Costumbrado, J., Hsu, C.Y., Kim, Y.H. (2012). Agarose gel electrophoresis for the separation of DNA fragments. J. Vis. Exp. (62):3923
- Findlay, D., Herries, D. G., Mathias, A. P., Rabin, B. R., and Ross, C. A. (1961). The active site and mechanism of action of bovine pancreatic ribonuclease. Nature 190: 781-784.
- Gersten, D. M., and Gabrielt, O. (1992). Staining for Enzymatic Activity after Gel Electrophoresis. Anal. Chem. 203: 181-186.
- Greiner-Stoeffele T, Grunow M, Hahn U. (1996). A general ribonuclease assay using methylene blue. Anal. Biochem. 240 (1):24-8
- Roth, J.S. and Milstein, S.W. (1952). A new assay method with P labeled yeast ribonucleic acid. J. Biol. Chem. 196(2):489-49
- Bechhofer, D. A. and Deutscher, M. P. (2019). Bacterial ribonucleases and their roles in RNA metabolism. Crit. Rev. Biochem. Mol. Biol.54(3): 242–300.
- Sato, S and Takenaka, S (2014). Highly Sensitive Nuclease Assays Based on Chemically Modified DNA or RNA. Sensors (Basel). 14(7): 12437–12450.
- Shapira, R. (1962). A spectrophotometric method for the measurement of ribonuclease activity. Analytical Biochemistry, 3, 308-320.
- Wagner, A.P., Lieselotte, M. C., and Wagner. P., (1983). A simple spectrophotometric method for the measurement of ribonuclease activity in biological fluids. J Biochem. Biophys. Methods. 8(4):291-297.