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.

Although ribonuclease inhibitor (RI), also known as ribonuclease inhibitor protein (RIP) and human placental ribonuclease inhibitor (hPRI), has been the most commonly used RNase inhibitor, it has limitations. RI binds only RNase A-type enzymes, functions under a narrow temperature range and requires DTT to prevent the release of active RNases. New SUPERase•In inhibits more RNases — RNase A, B, C, RNase T1 and RNase 1. SUPERase•In remains active up to 65C and has no DTT requirement. SUPERase•In gives a higher level of protection against RNA degradation than any other product. It's the ultimate RNase inhibitor.

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The Right Choice for Protecting Your RNA
SUPERase•In™ (patent pending) is now the clear choice among ribonuclease (RNase) inhibitors. SUPERase•In, like ribonuclease inhibitor (RI), also known as ribonuclease inhibitor protein (RIP) and human placental ribonuclease inhibitor (hPRI), is a protein inhibitor that works by noncovalent binding of RNases. Unlike RI, SUPERase•In does not require DTT to function, and it inhibits more RNases, at higher concentrations, under more reaction conditions than other RNase inhibitors. SUPERase•In can be used in any application where RNase contamination is a concern, and in any application where RI is now used. It does not interfere with enzymes such as RNA polymerase, reverse transcriptase or Taq polymerase. It is ideal for use in RT-PCR, cDNA synthesis, in vitro transcription and translation reactions, and preparation of RNase-free antibodies. Until now, hPRI has been the most widely used RI in molecular biology. This article discusses the advantages of using SUPERase•In instead.

Inhibit More RNases Than Any Other Inhibitor
RNase inhibitors are typically used during enzymatic reactions to protect RNA from RNase contamination introduced from one or more of several common, but diverse sources. hPRI has been the most widely used ribonuclease inhibitor over the past several decades. RI inhibits RNase A and its carbohydrate variants, RNases B and C. SUPERase•In not only inhibits these RNases, but it also inhibits RNase 1 and RNase T1. When considering where RNase contamination might originate, it becomes clear why you need to inhibit different types of RNases. RNase A, for example, is a common contaminant on laboratory equipment and supplies because it is present on human skin. It is used in large quantities for both plasmid and protein purification, and, along with RNase T1, it is used in ribonuclease protection assays. Bacterial RNases can affect experiments that include bacterial lysates, or proteins or DNA templates that are purified from overexpression in bacteria. Even commercial enzymes can be contaminated with trace amounts of RNases (all types). Environmental sources such as dust, ungloved hands, and contaminated solutions may also introduce many different types of RNase (1). Figure 1 shows the abilities of SUPERase•In and RI to inhibit different RNases. RI protects RNA from degradation by RNase A, but it had little or no ability to prevent degradation by RNase 1 or T1. In contrast, the RNA treated with SUPERase•In was protected from digestion by RNase A, RNase T1, and RNase 1.

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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. See also "What's in a Unit?".

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 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.

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.

Figure 3. Contaminating RNase Activity Measured by Denaturing PAGE and Autoradiography. Ribonuclease Inhibitors (200 U each) from supplier A (Panel A), supplier B (Panel B), and SUPERase•In™ (Panel C), were heat-inactivated at 67°C for 15 minutes under different conditions: 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™.