RNase Testing

RNase is common in living cells and breaks down RNA, which can contaminate reagents if it is not carefully controlled. Learn more about RNase testing for your custom reagent and how it can help protect both your reagents and your data.

Considerations & Limitations

Considerations and Limitations of RNase Testing
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
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 these 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.

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.

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