RNase Inactivation Reagents
Ribonuclease (RNase) are considered a nuisance in experiments involving RNA, because they degrade RNA and are not completely inactivated even by autoclaving. Once contaminated, RNase is not easily removed. Therefore, it is important not to bring RNase into the experimental system. Fujifilm Wako offers reagents to prevent RNase contamination, such as RNase removal spray and RNase inhibitors.
How Does RNase Contamination Occur?
According to Molecular Cloning, there are two main sources of contamination of exogenous RNases1).
Contamination in Buffers
Buffers are easily contaminated by bacteria and microorganisms, and once contaminated, RNase cannot be completely removed even if the buffer is autoclaved to kill bacteria and microorganisms. It is difficult to visually detect contamination, and buffers should be discarded if any contamination is suspected.
Contamination Through Pipettors
Caution is required when using the same pipettor in multiple experiments. In particular, if the pipettor had been used in plasmid extraction or ribonuclease protection assays, RNase could be contaminated from the body of the pipettor or its ejector, even if RNase-free tips are used.
How to prevent contamination of RNase
- Store stock solutions such as buffers in small portions whenever possible.
- Prepare buffers using RNase-free glassware or plasticware (disposable) and DEPC*-treated water, etc.
- Use instruments and reagents set aside for experiments involving RNA.
- Glassware should be heat treated at 300°C for 4 hours. Plasticware should be treated with DPEC, etc. Treatment with a commercially available RNase removal spray is also effective.
- RNase inhibitors may be added to in vitro transcription, RT-PCR, and cDNA synthesis reactions.
* DEPC (Diethylpyrocarbonate)
DEPC is a chemical modifier of histidine and tyrosine in proteins. It irreversibly inhibits the activity of RNase by modifying the histidine residue in its active center. DEPC is gradually hydrolyzed to CO2 and ethanol in aqueous solutions. It is rapidly hydrolyzed in the presence of Tris or other amines and cannot be used in buffers containing them. DEPC-treated water is prepared by first adding DEPC to water and then degrading DEPC by autoclaving. DEPC itself is suspected to be carcinogenic and should be handled with caution.
- Green, R. M. and Sambrook, J.: ”Molecular Cloning A Laboratory Manual, 4th ed.”, Cold Spring Harbor Laboratory Press, (2012).
- “Life Science Reagent Application Handbook” ed. by Tamura, T., Yodosha, Japan, (2009). (Japanese)
For research use or further manufacturing use only. Not for use in diagnostic procedures.
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