How to Prevent RNase Contamination in Proteinase K?

RNase is a ribonuclease, which is an enzyme that specifically breaks down RNA. Nucleases are present in every cell, and upon lysis during an extraction procedure, these enzymes come into contact with precious DNA and RNA, with RNases targeting RNA and DNases targeting DNA.

Preventing RNase contamination when working with proteinase K helps keep your nucleic acids intact during extraction procedures.

Sources of RNase contamination

In order to prevent RNase contamination, let’s first look at common places where contamination is likely to happen:

• Your hands or direct skin contact
• Pipette tips and tubes
• Environment and surfaces
• Reagents not specifically used for RNA or DNA extraction
• Samples

Preventing RNase contamination

Even if the reagents you bought are RNase-free, the listed channels of contamination could introduce RNase into your procedure. In order to minimize the risk of RNase contamination, here are a few best-practices when working with RNA or DNA.

• Wear gloves always. Don’t be afraid to change them often, especially after you have touched your phone, face or any other part of your skin or hair.
• Use RNase-free pipette tips and tubes. Autoclaved tips are not guaranteed to be free of RNase contamination.
• If possible, designate a special set of pipettes for extraction.
• Designate special glass and plastic ware solely for extraction
• Designate a special stock of reagents solely for your extraction procedures. These reagents are not to be used for anything else but extraction.
• Aliquot your extraction reagents and discard them after you have used them.
• Clean working surfaces thoroughly.
• Properly decontaminate all labware such as glassware and plastics. Heat glassware at 180-250°C for at least four hours.

References

Blink - UCSD. (2019, January 29). Biosafety: Decontamination Methods for Laboratory Use. Retrieved March 25, 2020, from https://blink.ucsd.edu/safety/research-lab/biosafety/decontamination/index.html#Heat-sterilization-(wet-or-dry)

Hossain, S. T., Malhotra, A., & Deutscher, M. P. (2016). How RNase R Degrades Structured RNA Role of the Helicase activity and the S1 Domain. Journal of Biological Chemistry, 291(15), 7877-7887.

Kanwar, S. S., Mishra, P., Meena, K. R., Gupta, S., & Kumar, R. (2016). Ribonucleases and their Applications. Journal of Advanced Biotechnology and Bioengineering, 4(1), 17-26.

Krutova, Marcela. (2017). What is the role of Proteinase K for Viral RNA extraction? Retrieved from: https://www.researchgate.net/post/What_is_the_role_of_Proteinase_K_for_Viral_RNA_extraction/589df672eeae39977d534023/citation/download.

McGookin, R. (1984). RNA extraction by the proteinase K method. In Nucleic Acids (pp. 109-112). Humana Press.

Open WetWare . (2009, January 30). Avoiding RNase contamination. Retrieved March 25, 2020, from https://openwetware.org/wiki/Avoiding_RNase_contam...