The Right Choice for Protecting Your RNA
Inhibit More RNases Than Any Other Inhibitor
Figure 1. SUPERase•In vs. Human Placental Ribonuclease Inhibitor (RI). A 32P-labeled RNA probe was incubated for 30 minutes at 37°C in the presence of the indicated nucleases and either SUPERase•In or human placental ribonuclease inhibitor protein (RI). Both the SUPERase•In and the RI were added at a concentration of 1 U/µl.
SUPERase•In Is Active Over a Broader Range of Conditions Than RI
Without DTT, RI May Release Active RNase -- SUPERase•In Won't.
Figure 2. RNase Activity In Ribonuclease Inhibitor Preparations. SUPERase•In and 3 RNase Inhibitors from other suppliers were analyzed for the presence of latent RNases in a microplate assay using a SpectraMAX Gemini XS spectrofluorometer. To detect latent RNase activity, the inhibitors were incubated at 67°C for 15 minutes under reducing and oxidizing conditions to release any bound contaminating RNases. 200 U of each inhibitor was then tested with a fluor/quenched RNA substrate using the RNaseAlert® assay. Reactions were monitored in real-time at 37°C over 60 minutes in 5-minute increments. Relative fluorescence units (RFU) generated during incubation of the RNaseAlert substrate with RNase Inhibitors was then plotted.
Detecting Latent RNases Associated With RNase Inhibitors
The data in Figure 2 address whether pre-heating of RIs releases latent RNase activity associated with the inhibitors. RNase activity was detected in 2 out of 3 of the other suppliers' RIs tested. Preheating SUPERase•In, in contrast, caused no detectable release of RNase, as exhibited by the lack of signal fluorescence elevation over background.
The data in Figure 2 were confirmed by incubation of RNase Inhibitors with a 32P-radiolabeled RNA probe followed by PAGE and autoradiography. Results are presented in Figure 3. In contrast to the other RNase Inhibitors, SUPERase•In showed no RNase activity under any of the conditions tested. The highest level of latent RNase contaminants in the other RIs was observed in the absence of DTT and/or in the presence of oxidized glutathione. The results also confirm that the RIs from some commercial sources may have bound RNase contaminants associated with them. In the presence of DTT, degradation of the RNA substrate by latent RNase present in RI from supplier A was observed during overnight incubation, but not after 1 hour incubation. In the case of RI from supplier B, some degradation of RNA was observed in the presence of DTT even after only 1 hour of incubation. In the absence of DTT, RNase activity was sufficiently high to partially degrade the probe after only 1 hour. Complete degradation occurred during overnight incubation.
Heating of RIs from both suppliers A and B in the presence of oxidized glutathione caused an even greater release of latent RNase such that most of the probe was degraded after 1 hour incubation. In contrast, Ambion's SUPERase•In showed no presence of latent RNases under all conditions tested.
- in storage buffer containing 8 mM DTT (20 mM HEPES-KOH pH 7.6, 50 mM KCl, 8 mM DTT, 50% glycerol),
- in storage buffer with DTT depleted by dialysis,
- in storage buffer containing DTT together with 5 mM oxidized glutathione,
- in storage buffer minus DTT, with 5 mM oxidized glutathione.
Each heat-treated RNase Inhibitor (200 U) was then incubated at 37°C in duplicate 20 µl reactions containing 2 µg of radiolabeled RNA probe, 50 mM Tris-HCl (pH 7.5), 50 mM KCl, 1 mM EDTA, for 1 hour and overnight. The radiolabeled RNA probe was separated on a 5% acrylamide/8M urea gel and detected by autoradiography (30 min. exposure). One-hour reactions are presented for supplier's A and B, and the overnight reaction is presented for SUPERase•In.
SUPERase•In is the Clear Choice
- Linn, S., Lloyd R., Roberts, R. (1993) Nucleases. Cold Spring Harbor Laboratory Press. Plainview, New York.
The traditional unit assay for ribonuclease inhibitors has been the cyclic CMP assay. While this assay does demonstrate inhibition of RNase A, it is limited in that it provides only an indirect measure of RNA degradation. Because cCMP is an artificial substrate for RNase, its hydrolysis does not always correlate with actual RNA degradation. In addition, this assay can only be used for RNase A-type ribonucleases and their inhibitors because cCMP is not a substrate for many other ribonucleases.
Ambion has recently developed a new unit assay for ribonuclease inhibitors. It is a functional assay that directly measures the ability of the inhibitor to block RNA degradation. A radiolabeled RNA is exposed to various RNases with and without the inhibitor. The results are analyzed on a polyacrylamide gel to determine the degree to which RNA degradation is inhibited. This functional assay provides direct information about the inhibitor's ability to block RNA degradation.