Researchers are usually trained in RNA isolation and analysis methods by one another or by technical manuals. Experimental procedures are often not questioned and quickly become dogma. Furthermore, it is difficult to find literature to document the "facts" taught by mentors and technical manuals. One of these potential myths is the use of DEPC treatment to make solutions RNase-free. We have systematically investigated some of the DEPC fables, and the results are discussed below. Watch upcoming issues of TechNotes in which we will put other RNA "truths" to the test.
1. Autoclaving is not effective at eliminating RNase in solution because the RNases simply renature as the solution cools.
FALSE, but... Autoclaving alone does indeed inactivate a substantial amount of RNase A (Figure 1). Various concentrations of RNase A were added to PBS and autoclaved. Aliquots of each solution were mixed with a 304 base 32P-labeled RNA probe and incubated at 37°C for one hour, followed by electrophoresis and exposure to film. Without autoclaving, the probe begins to degrade at an RNase concentration of 100 pg/ml. Autoclaving inactivates enough of the RNase A to protect the probe from degradation up to a concentration of 1 µg/ml. Note that only a portion of the RNase is inactivated by autoclaving, otherwise the RNA probe would remain intact at any RNase concentration. Autoclaving alone may be sufficient to eliminate enough RNase for some applications. However, since neither the extent of RNase contamination nor at what RNase concentration the assay is sensitive is known, DEPC should be used as an added precaution. Also note that these experiments were only performed on RNase A and may not hold true for other RNases.
Figure 1. Effect of Autoclaving on RNase Activity. Various concentrations of RNase A were added to PBS and autoclaved for 25 minutes. 1 µl of each solution was mixed with 1 ng of a 5 x 104 cpm RNA probe which was 304 bases long and incubated at 37°C for one hour. 5 µl of the reaction was assessed on a 5% acrylamide/8 M urea gel and exposed to film for 5 hours with an intensifying screen.
2. Autoclaving inactivates DEPC
TRUE. Autoclaving does inactivate DEPC by causing hydrolysis of diethylpyrocarbonate. CO2 and EtOH are released as reaction by-products. DEPC has a half-life of approximately 30 minutes in water, and at a DEPC concentration of 0.1%, solutions autoclaved for 15 minutes/liter can be assumed to be DEPC-free.
3. Autoclaving DEPC-containing solutions should be of sufficient duration to rid the solution of any smell
FALSE. A faint EtOH smell may linger after autoclaving, but more commonly a sweet, fruity smell is observed. This is caused by the EtOH by-product combining with trace carboxylic acid contaminates and forming volatile esters. It does not mean that trace DEPC remains in the solution.
4. Solutions containing Tris cannot be treated with DEPC
TRUE. Tris contains an amino group which "sops up" DEPC and makes it unavailable to inactivate RNase (Figure 2). 1 M solutions of Tris, MOPS, HEPES and PBS were prepared, and 0.1% or 1% DEPC was added to each. One µg/ml RNase A was also added to each solution. The solutions were autoclaved and aliquots of each solution were mixed with a 304 base 32P-labeled RNA probe and incubated at 37°C for one hour. Probe integrity was assessed by electrophoresis and exposure to film. Tris and HEPES do indeed make DEPC unavailable to inactivate RNase at a DEPC concentration of 0.1% (recommended by most protocols). However, 1% DEPC is sufficient to overcome this effect. When 1M MOPS and PBS are treated with DEPC, the DEPC remains available to inactive RNase at both concentrations (0.1% and 1%). It would be impossible to predict the different interactions of DEPC with all molecular biology reagents. The most cautious approach for making RNase-free solutions would be to mix molecular biology grade powdered reagents up in DEPC-treated water. Alternatively, many pre-made nuclease-free solutions can be purchased from Thermo Fisher Scientific and other companies.
Figure 2. Effects of DEPC Treatment of Various Buffers. One µg/ml RNase A was added to various buffers along with 0.1% or 1% DEPC. Solutions were vigorously shaken for 1 minute, incubated for 1 hour at room temperature and autoclaved for 25 minutes. 1 µl of each solution was mixed with 1 ng of a 5 x 104 cpm 304 nt RNA probe and incubated at 37°C for one hour. 5 µl of the reaction was assessed on a 5% acrylamide/8 M urea gel and exposed overnight to film.
5. 0.1% DEPC is sufficient to inhibit any amount of RNase in a solution
FALSE, but... The amount of DEPC required to inactivate RNase increases as the amount of contaminating RNase in a solution increases (Figure 3). 100, 500, and 1000 ng/ml RNase A was added to water followed by various amounts of DEPC. The solutions were autoclaved, and aliquots of each solution were mixed with a 304 base 32P labeled RNA probe and incubated at 37°C for one hour followed by electrophoresis and exposure to film. Untreated solutions or those treated with 0.01% DEPC could inactivate 100 ng/ml RNase A. When the RNase concentration was increased to 500 ng/ml, the DEPC concentration was insufficient to inactivate the RNase, and the probe degraded. Increasing the DEPC concentration to 0.1% protects the probe from RNase A up to 500 ng/ml and a 1% DEPC solution inactivates RNase A at a concentration of 1000 ng/ml. 0.1% DEPC is probably adequate to inactivate most RNase contamination from environmental sources and from laboratory procedures such as ribonuclease protection assays and plasmid preparations in which larger amounts of RNase are used.
Figure 3. Effects of Varying Percentages of DEPC on Increasing Concentrations of RNase. Various concentrations of RNase A were added to aliquots of water, shaken vigorously for one minute, incubated for one hour at room temperature and autoclaved for 25 minutes. 1 µl of each solution was mixed with 1 ng of a 5 x 104 cpm RNA 304 nt probe and incubated at 37°C for one hour. 5 µl of the reaction was assessed on a 5% acrylamide/8 M urea gel and exposed overnight to film.
6. If 0.1% DEPC works well to inhibit RNase, 1% should work even better
TRUE, but... Increasing DEPC concentrations inactivate increasing amounts of RNase A contamination (Figure 3). However, it is also true that high levels of residual DEPC or DEPC by-products in a solution can inhibit some enzymatic reactions or chemically alter (carboxymethylate) RNA. It has been documented that DEPC byproducts in RNA samples can inhibit in vitro translation reactions (Winkler, unpublished results). For this study, we tested transcription reactions for DEPC inhibition effects. Template DNA was dried to completion in a vacuum centrifuge and resuspended in 0.01%, 0.1% or 1% DEPC-treated water. Duplicate MAXIscript™ transcription reactions were assembled using 32P-UTP, and the same concentration of DEPC treated water bought up the volume. The reactions were incubated and percent incorporation was assessed via TCA precipitation. The average percent incorporation were as follows:
|% DEPC in reaction||% incorporation|
The above data indicate that increasing amounts of DEPC increasingly inhibit transcription. Again, 0.1% DEPC is probably sufficient to inhibit most RNases with minimal effect on reactions. If DEPC is suspect in inhibiting reactions, high quality (MilliQÌ) or autoclaved water can probably be substituted into the reaction. Water can be tested using Ambion's RNaseAlert™ Kit or see Technical Bulletin #166, Nuclease and Protease Testing: Laboratory and Commercial Considerations, which describes an RNase testing protocol similar to that used in this study.