Decorative molecules with a red background

Ribonucleases—or RNases—play important roles in nucleic acid metabolism, are found in both prokaryotes and eukaryotes, and in practically every cell type. The human body uses RNases to defend against invading microorganisms by secreting these enzymes in fluids such as tears, saliva, mucus, and perspiration. RNases are found in flaked skin, on hair that may fall onto a bench, and in pet hair that may cling to clothing. The primary source of RNases within most environments, however, is microorganisms—namely, bacteria and fungi. In this article, we discuss how to avoid, detect, and inhibit RNase when working with RNA.

Working with RNA RNA basics

Navigate the basics of RNase control:

What are RNases?

RNases are enzymes that play an important role in RNA degradation. RNases have several cysteine residues that form numerous intramolecular disulfide bonds—the robust nature of these enzymes makes them refractory to many methods of decontamination, and strong chemical methods are often required to eliminate RNases from surfaces and solutions.

What does RNase do?

RNases catalyze cleavage on RNA, which contributes to the degradation of RNA. They break the bonds between nucleotides, breaking RNA into smaller components and allowing access to other enzymes. RNases are usually considered contaminants in labs where researchers work with RNA because it lessens RNA integrity and sample quality.

RNases in the lab

To protect from RNases in lab, RNA samples must be properly stored, buffers and solutions routinely detected for RNase contamination, and RNases inhibited or removed from lab spaces with the use of RNase decontamination solution and RNase inhibitors.

Why is sample storage is critical for RNA samples?

The presence of just trace amounts of RNase can compromise RNA integrity even if the samples are stored frozen in an aqueous environment. The following are best practices for storing RNA samples:

For short-term storage: RNA samples can be resuspended in RNase-free water (with 0.1 mM EDTA) or TE buffer (10 mM Tris, 1 mM EDTA) and stored at –80°C. Using a buffer solution that contains a chelating agent is a better way to store RNA. Chelation of divalent cations such as Mg2+ and Ca2+ will prevent heat-induced strand scission (RNA can be chemically cleaved when heated in the presence of Mg2+).

For long-term storage: Perform a salt/alcohol precipitation on pre-aliquoted sample and store the nucleic acid as a precipitate in this solution at –20°C. The low temperature and the presence of alcohol inhibit all enzymatic activity. The lower than neutral pH (due to the presence of sodium acetate or ammonium acetate) also helps stabilize the RNA. Note that the RNA will have to be centrifuged out of this solution prior to any downstream application.

Read more about working with RNA

Sources of RNase contamination

Common sources of RNase contamination include lab benches, pipettors, glassware, tubes, tips, and electrophoresis equipment. When working with RNA, it’s important to ensure that lab plastic consumables, such as microcentrifuge and PCR tubes and pipette tips, are RNase-free. All lab surfaces, such as benchtops, centrifuges, and electrophoresis equipment should be presumed contaminated with RNases since they are exposed to environmental contaminants. Sources of RNase contamination on lab surfaces often include bacterial and fungal spores and dead cells shed from human skin (e.g., hands).

Due to the ubiquitous nature of RNases, water and buffers used in molecular biology applications can be frequent sources of RNase contamination. DEPC treatment is the most common method used to inactivate RNases in water and buffers. However, certain reagents such as Tris cannot be DEPC-treated.

RNA strand scission

Though RNase contamination is commonly suspected whenever RNA degradation is observed, RNA molecules can also undergo strand scission when heated in the presence of divalent cations such as Mg2+ or Ca2+ at >80°C for five minutes or more. Thus, a chelating agent should be present whenever there is a requirement for heating RNA.

Ambion-scientist recommended schedules for RNase control

Ambion scientists recommend the following schedule for RNase contamination control:

  • Daily
    • Use RNase-free buffers and reagents
    • Use RNase-free consumables, such as microcentrifuge tubes and pipette tips
    • Use Ribonuclease Inhibitor Protein and/or ANTI-RNase in reactions such as in vitro transcription and RT-PCR
  • Weekly
    • Thoroughly clean lab benchtops, pipettors, and tube racks
  • Monthly
    • Test water sources for RNases
  • As Needed
    • Test bench-prepared reagents for RNases
    • Clean electrophoresis equipment prior to use with RNA
    • Use barrier or filter pipette tips when performing procedures that incorporate the use of RNase

Explore RNase-free buffers and reagents
Explore RNase-free tubes and pipette tips

RNA sample storage

Why is sample storage is critical for RNA samples?

The presence of just trace amounts of RNase can compromise RNA integrity even if the samples are stored frozen in an aqueous environment. The following are best practices for storing RNA samples:

For short-term storage: RNA samples can be resuspended in RNase-free water (with 0.1 mM EDTA) or TE buffer (10 mM Tris, 1 mM EDTA) and stored at –80°C. Using a buffer solution that contains a chelating agent is a better way to store RNA. Chelation of divalent cations such as Mg2+ and Ca2+ will prevent heat-induced strand scission (RNA can be chemically cleaved when heated in the presence of Mg2+).

For long-term storage: Perform a salt/alcohol precipitation on pre-aliquoted sample and store the nucleic acid as a precipitate in this solution at –20°C. The low temperature and the presence of alcohol inhibit all enzymatic activity. The lower than neutral pH (due to the presence of sodium acetate or ammonium acetate) also helps stabilize the RNA. Note that the RNA will have to be centrifuged out of this solution prior to any downstream application.

Read more about working with RNA

Contaminants

Sources of RNase contamination

Common sources of RNase contamination include lab benches, pipettors, glassware, tubes, tips, and electrophoresis equipment. When working with RNA, it’s important to ensure that lab plastic consumables, such as microcentrifuge and PCR tubes and pipette tips, are RNase-free. All lab surfaces, such as benchtops, centrifuges, and electrophoresis equipment should be presumed contaminated with RNases since they are exposed to environmental contaminants. Sources of RNase contamination on lab surfaces often include bacterial and fungal spores and dead cells shed from human skin (e.g., hands).

Due to the ubiquitous nature of RNases, water and buffers used in molecular biology applications can be frequent sources of RNase contamination. DEPC treatment is the most common method used to inactivate RNases in water and buffers. However, certain reagents such as Tris cannot be DEPC-treated.

RNA strand scission

Though RNase contamination is commonly suspected whenever RNA degradation is observed, RNA molecules can also undergo strand scission when heated in the presence of divalent cations such as Mg2+ or Ca2+ at >80°C for five minutes or more. Thus, a chelating agent should be present whenever there is a requirement for heating RNA.

Facility hygiene scheduling

Ambion-scientist recommended schedules for RNase control

Ambion scientists recommend the following schedule for RNase contamination control:

  • Daily
    • Use RNase-free buffers and reagents
    • Use RNase-free consumables, such as microcentrifuge tubes and pipette tips
    • Use Ribonuclease Inhibitor Protein and/or ANTI-RNase in reactions such as in vitro transcription and RT-PCR
  • Weekly
    • Thoroughly clean lab benchtops, pipettors, and tube racks
  • Monthly
    • Test water sources for RNases
  • As Needed
    • Test bench-prepared reagents for RNases
    • Clean electrophoresis equipment prior to use with RNA
    • Use barrier or filter pipette tips when performing procedures that incorporate the use of RNase

Explore RNase-free buffers and reagents
Explore RNase-free tubes and pipette tips

RNA preservation and RNase removal for high quality RNA

There are a variety of ways to preserve RNA and protect samples from RNase contamination. For one, RNA stabilization via storage in RNA stabilization solution helps preserve the integrity of RNA. Secondly, RNase detection should occur in a cyclical and as-needed basis, such as water testing and reagent testing. Thirdly, RNase removal to aid in RNase control in labs where RNA samples are precious. RNase removal methods include RNase inhibition and RNase decontamination.

NOTE: It is highly recommended that a dedicated space be set aside for procedures that require RNase treatment to avoid inadvertent exposure of RNA samples to RNase.

Stabilize and preserve the integrity of your RNA samples

RNA stabilization icon

RNA stabilization reagents are available to lengthen the usage of RNA samples by stabilizing RNA molecules and preserving RNA integrity.

Explore RNA stabilizers

Test water and buffers regularly for RNase activity

RNase detection icon

Constant RNase monitoring is necessary for researchers who work with RNA. It’s important to keep a regular schedule for detecting RNase contamination. Our scientist-approved schedule for maintaining RNase-free lab spaces is a solid start for RNase control.

Learn more about RNase detection

RNase inhibitor proteins
 

RNase inhibitors icon

The traditional method for combating RNases in enzymatic reactions such as in vitro transcription, reverse transcription, and translation is to use a ribonuclease inhibitor. This protein is an inhibitor of only the RNase A family of ribonucleases, which includes RNases A, B, and C.

Learn more about RNA inhibitors

Prevent RNase contamination by using decontamination supplies

RNase decontamination icon

Discussed above are the many sources of RNase contaminants. Because RNases exhibit such a common presence in routine lab research, it is essential to develop a program of routine RNase decontamination. RNase decontamination reagents are available in spray bottle and towelette form for convenience.

Learn more about RNase decontamination

 

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