| SUPERase•In™ - The Ultimate RNase Inhibitor 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 noncovalently binding RNases. Unlike RI, SUPERase•In doesn't 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 example, in RT-PCR, cDNA synthesis, in vitro transcription and translation reactions, and preparation of RNase-free antibodies. Until now, placental ribonuclease inhibitor has been the most widely used RNase inhibitor in molecular biology. This article discusses the shortcomings of RIP, and the advantages of using SUPERase•In instead. by Tiffany Smith and Lori Martin RNase inhibitors are typically used to protect RNA during enzymatic reactions from RNase contamination introduced from one or more of several common, but diverse sources. RI has been the most widely used ribonuclease inhibitor used for this purpose 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 RNases 1 and T1. When you consider 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 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, such as RNase 1, often plague 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 has 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 RNases A, T1, and RNase 1. | | | Figure 1. Inhibition of Ribonucleases by SUPERase•In™ vs. Placental Ribonuclease Inhibitor. 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 ribonuclease inhibitor protein (RIP). Both the SUPERase•In and the RI (indicated here as RIP) were added at a concentration of 1 U/µ1. | SUPERase•In Is More Robust Than RI Because it's impossible to know how much RNase might be inadvertently introduced into an experiment, we've addressed how much RNase both RI and SUPERase•In can protect against. When RI is challenged with high concentrations of RNase A, it fails. SUPERase•In, however, can inactivate up to 300 pg of RNase A. The experiment in Figure 2 shows the addition of equivalent cCMP assay units (1 cCMP unit/µl of reaction) of either RI or SUPERase•In to a 32P-labeled RNA transcript. RNA probe integrity was then challenged with increasing amounts of RNase A. After 30 minutes incubation with RNase, the samples were analyzed on a denaturing acrylamide gel and exposed to film. In the presence of only 20 pg of RNase A, some RNA probe degradation already starts to become visible in the sample containing RI. SUPERase•In, however, protects the probe from degradation at concentrations of RNase as high 200-300 pg RNase A. At this concentration of RNase A, the protective effect of RI is negligible and the labeled RNA is barely visible. | | | Figure 2. Inhibition of High Concentrations of Ribonuclease A. A 32P-labeled RNA probe was incubated for 30 minutes in the presence of increasing amounts of RNase A and either SUPERase•In or ribonuclease inhibitor (RIP). The samples were then analyzed on a denaturing acrylamide gel and exposed to film. | In the Absence of DTT, RI May Release Active RNase - SUPERase•In Won't. Most protocols recommend using RI in a reducing environment (typically 1 mM DTT). Without DTT, bound RNase can be released in an active form. In fact, if RI becomes oxidized, RNase bound to RI can be released into your experimental sample and degrade RNA. Freeze/thaw and exposing a sample to air can both result in oxidation and inactivation of DTT. SUPERase•In has no DTT requirement, but it is also fully functional in DTT concentrations as high as 200 mM DTT. SUPERase•In Is Active Over a Broader Range of Conditions Than RI Because of its robust interaction with RNase, SUPERase•In remains active over a broad range of conditions, providing flexibility in experimental design. In addition to SUPERase•In's activity in the absence (or presence) of DTT, one of the most important differences between RI and SUPERase•In is the effect of temperature on their activity. SUPERase•In will effectively inhibit RNases from 4°C to 65°C, whereas RI loses activity at temperatures above 50°C. SUPERase•In's broad functional temperature range is useful when it is necessary to adjust reaction temperatures to resolve problematic transcription and reverse transcription reactions. Also, SUPERase•In is effective from pH 5.5 to 8.5; the optimal pH range for RI is 7-8. Both RI and SUPERase•In tolerate common detergents up to about 3%, but SUPERase•In remains effective in the presence of higher concentrations of the denaturants guanidinium thiocyanate (up to 3 M) and urea (up to 6 M). If effectiveness, flexibility, and quality are important factors in your choice of RNase inhibitor, then choose SUPERase•In. It is the most effective RNase inhibitor available, providing the highest level of protection against the most RNases, in the most diverse conditions. 1. Linn, S., Lloyd R., Roberts, R. (1993) Nucleases. Cold Spring Harbor Laboratory Press. Plainview, New York. The Truth About Ribonuclease Inhibitor Protein (RI, hPRI or RIP) One of the most commonly used tools to combat RNase contamination is placental ribonuclease inhibitor or RI, a protein that binds RNase molecules. While this protein can be used to block RNase contamination during in vitro transcription, its function is often misunderstood. RI does not destroy RNases. The mode of inhibition is noncompetitive; the inhibitor tightly binds RNase A type enzymes only (including RNases A, B and C) in a 1:1 ratio. To maintain activity, RNase inhibitor is generally reported to require a minimum DTT concentration of 1 mM. RNase inhibitor proteins complex with active RNases, and the denaturation or oxidation of the inhibitor can result in the release of active ribonuclease. Care should be taken to avoid any procedure, such as heat denaturation or oxidizing conditions, that might result in the release of active RNases. Ambion's new ribonuclease inhibitor, SUPERase•In™, provides a more robust alternative to traditional ribonuclease inhibitors.
What’s in a Unit?
Most enzymes and proteins are sold based upon their unit activity. Have you ever wondered what that unit means, or do you just follow the manufacturer’s guideline for recommended use? Looking closely at not only the manufacturer’s unit definition, but more importantly at their unit assay, generates a better understanding of how the product will function in your specific application. Unfortunately, more often than not, the attempt to apply an established unit assay to your own system leads to headache and frustration because unit assays are designed to quantitate a specific function of the product. Such is the case for ribonuclease inhibitors. The traditional unit assay for ribonuclease inhibitors has been the cCMP 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 as cCMP is not a substrate for many other ribonucleases. Ambion has recently developed a new unit assay for ribonuclease inhibitors. It is a true 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. |