Setting up for RNA work- additional considerations

Written By jeremy hogenbaum
a scientific image RNA purification work

RNases are everywhere and are difficult to completely get rid of, however managing RNAse contamination is critical to any setting in which it is paramount to maintain the integrity of RNA.¹ There is plenty of instruction on general considerations. This post is meant to supplement the plethora of guides out there and is not meant to be a standalone or comprehensive list.²⁻⁴

Good air quality is absolutely essential

  • RNases are common to almost all living things, especially humans.⁵ Major components in dust are human skin cells and hair, so, by extension, dust can contain RNases.⁶ It is therefore important to maintain adequate air quality to minimize the possibility of RNase contamination. Many clean benches maintain Iso 5 (formerly class 100) filtration with positive pressure. ⁷,⁸

  • It is of important note that laminar flow hoods are not biosafety cabinets (BSC), if you are operating with BSL-2 material make sure to be using a BSC of the appropriate class.⁹

UV is a powerful ally

  • The proper combination of UV wavelengths can decontaminate surfaces from RNases in under 5 minutes. ¹⁰ Make sure to not expose you (and others) to harmful UV light.¹¹

Packaging, make the most of thorough industry decontamination processes

  • RNase free is a designation that is validated by the supplier (it still is a good idea to test for contamination, however). Use this to your advantage, items in uncompromised packaging are more likely to be RNase free than manually cleaned items. Open your consumables without contacting the interior of the packaging or the item itself, eliminate (or at least minimize) exposure to poor quality air.

Isolation

  • Maintaining cleanliness decreases chances of inadequate cleaning procedures impacting your experiment in the long run. Lower burdens make it easier to clean down acceptable levels. Keep your equipment for RNA work together and in a decontaminated environment and don’t use this equipment for anything else.¹²

Beware of opening caps

  • Often RNA work uses smaller sample tubes with snap caps. Be exceptionally careful that you don’t touch the internal portion (including the sidewalls since these will be going back into the tube). This should be a part of standard aseptic technique but pay extra attention to your excess glove material and be cautious.

Consider passivation of your steel equipment

  • Products that are designed to remove RNases from surfaces tend to be corrosive, especially if not properly rinsed away.¹³ Consider passivating your steel in your RNA work environment to help mitigate damage. ¹⁴ It will also improve the efficacy of your surface cleaning methods, as there will be fewer pits and micro-abrasions on your metal work surfaces. ¹⁵

Other material management

  • Products that are designed to remove RNases often suggest rinsing away solution with RNase free water and wiping up with an absorbent material. ¹⁶ Well, you just spent all that time removing RNases from your work surface and now you are going to wipe it up with an uncertified, RNase-questionable product. It is not ideal, but at the very least it is good to use lint-free absorbent material that has been set aside specifically for RNA work . ¹⁷ If opening a new box make sure to use an RNase-free gloved hand to pull out the first wipe, try to keep these wipes in a quality air environment (but this often not practical due to limited work space), if you can’t, consider not using the exposed wipe but instead use the next one (or the one after that for good measure). Save the unused wipes for other cleaning tasks in the lab.

Making Tris buffers

  • DEPC is a great way to make rinse water but its method of RNase activation makes it incompatible with buffers that have primary amines. ¹⁸ At the very least, keep a separate Tris stock that is only used with RNase decontaminated tools (preferably in quality air) and reconstituting with RNase free water… or just purchase these buffers from a reputable company. ¹⁹

Remember, a lost experiment due to RNase contamination often will cost you more than the time to properly prepare your RNA workstation. Take your time, be careful, and don’t let RNases get the better of you!

 
 

References:


1. Robb, B. G.; Hornblower, B. Avoiding Ribonuclease Contamination https://www.biocompare.com/Bench-Tips/169724-Avoiding-Ribonuclease-Contamination/  (accessed Sep 11, 2020). 
2. McAfferty, S. A Beginners guide to working with RNA Part 1: Background https://edinburghcrf.wordpress.com/2018/10/25/a-beginners-guide-to-working-with-rna-part-1-background/  (accessed Sep 11, 2020). 
3. Lab Depot, I. How to Remove RNase or DNase from Plastic Containers https://www.labdepotinc.com/articles/rnase-and-dnase-removal.html  (accessed Sep 11, 2020). 
4. New England Biolabs Inc. Avoiding Ribonuclease Contamination https://www.neb.com/tools-and-resources/usage-guidelines/avoiding-ribonuclease-contamination  (accessed Sep 11, 2020). 
5. Oswald, N. RNases: Their baddie super-powers explained (and how you can defeat them) https://bitesizebio.com/13528/rnases-their-baddie-super-powers-explained-and-how-you-can-defeat-them/  (accessed Sep 11, 2020).
6. Pelley, J. Tracing the Chemistry of Household Dust. c&en 2017, 95 (7). 
7. Arrouart, D. Understanding Cleanroom Classifications (ISO 8, ISO 7, ISO 6, ISO 5). 
8. Genovesi, L. PCR Enclosures: Essential for Improving PCR Accuracy https://www.labcompare.com/10-Featured-Articles/168593-PCR-Enclosures-Essential-for-Improving-PCR-Accuracy/  (accessed Sep 11, 2020). 
9. Air Science. Vertical Laminar Flow Hoods vs Biological Safety Cabinents https://www.airscience.com/briefs/briefnum/9/vertical-laminar-vs-bsc  (accessed Sep 11, 2020). 
10. Phoseon Technology. Controlling Ribonuclease (RNase) with High Irradiance UV LED Light Engines https://offers.the-scientist.com/hubfs/Phoseon RNase White Paper/Phoseon White Paper.pdf (accessed Sep 11, 2020). 
11. McLeod, V. Avoiding UV Light Hazards in the Lab https://www.labmanager.com/lab-health-and-safety/avoiding-uv-light-hazards-in-the-lab-1098  (accessed Sep 11, 2020). 
12. Oswald, N. 10 Ways to Work RNase Free https://bitesizebio.com/163/10-ways-to-work-rnase-free/  (accessed Sep 11, 2020).
13. Ambion. RNaseZap https://www.niehs.nih.gov/research/resources/assets/docs/instrument_cleaning_rnase_decontamination_508.pdf  (accessed Sep 11, 2020). 
14. Thermo Scientific. 1300 Series A2 Class II, Type A2 Biological Safety Cabinet Operating Manual 7021355 Rev. 10 https://physiology.case.edu/media/eq_manuals/eq_manual_forma_1300_seriesa2_class_2_type_a2_biological_safety_cabinet.pdf  (accessed Sep 11, 2020). 
15. CELCO. Passivation For The Pharmaceutical Industry https://www.celcoinc.com/industries/pharmaceutical/  (accessed Sep 11, 2020). 
16. Thermofisher Scientific. RNaseZapTM RNase Decontamination Wipes https://www.thermofisher.com/order/catalog/product/AM9786#/AM9786  (accessed Sep 11, 2020). 
17. HIMEDIA. HiPurA 96 Total RNA Purification Kit http://himedialabs.com/TD/MB612.pdf  (accessed Sep 11, 2020). 
18. Doronina, V. DEPC: The Wicked Witch of RNA https://bitesizebio.com/9429/depc-the-wicked-witch-of-rna/  (accessed Sep 11, 2020). 
19. Thermofisher Scientific. RNase and DEPC Treatment: Fact or Laboratory Myth https://www.thermofisher.com/us/en/home/references/ambion-tech-support/nuclease-enzymes/tech-notes/rnase-and-depc-treatment.html  (accessed Sep 11, 2020).
jeremy hogenbaum
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