Combat RNase Contamination in the Lab
|Trace amounts of ribonuclease (RNase) contamination can sabotage experiments. Since RNases are ubiquitous in the laboratory environment--on your skin, in the air, on anything touched by bare hands or on anything left open to the air--it is important to make an effort to prevent or eliminate RNase contamination. However, determining which piece of equipment or solution is responsible for RNase contamination can be frustrating and time-consuming. Ambion provides a complete arsenal of products to help prevent, detect, and eliminate RNase contamination in molecular biology experiments.|
SUPERase•In™ RNase Inhibitor (patent pending) is a protein-based inhibitor of nonhuman origin that noncovalently binds and inhibits the most common and troublesome RNases including RNase A, B, C, 1 and T1. SUPERase•In can be used in any application where RNase contamination could be problematic. It is ideal for in vitro transcription and translation, cDNA synthesis, RT-PCR, and preparation of RNase-free antibodies. Because it inhibits a broader range of RNases than traditional RNase inhibitors, SUPERase•In is the most effective RNase inhibitor available, providing a higher level of protection against degradation (Figure 1).
Figure 1. SUPERase•In™ vs. 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 RI. Both the SUPERase•In and the RI were added at a concentration of 1 U/µl.
The most widely used RNase inhibitor is placental ribonuclease inhibitor (RI), sold as Ribonuclease Inhibitor Protein. SUPERase•In is distinct from RI in that it has a more robust interaction with RNases, does not require DTT for activity, and inhibits a broader range of RNases. While placental ribonuclease inhibitor is effective only against RNase A-type enzymes (e.g., RNase A, B, and C), SUPERase•In also inhibits RNase I and T1. SUPERase•In does not interfere with other enzymes such as RNA polymerases, reverse transcriptase or Taq polymerase. Additionally, SUPERase•In is active up to 65°C and at pH 5.5 8.5.
RNase-free Buffers and Reagents
It is essential that any buffer or reagent added to purified RNA be free of RNase contamination. A simple alternative to the tedious preparation of RNase-free solutions is to purchase certified RNase-free water, buffers, and reagents such as those offered by Ambion. After carefully isolating the RNA, it is crucial that the pellet be resuspended and stored in an RNase-free solution. Ambion's molecular biology reagents are tested under extremely stringent conditions for consistent lot-to-lot quality. Our quality control philosophy is to subject our reagent products to conditions 10-100 times more stringent than those experienced under normal use.
If you need to detect RNases, then RNaseAlert® is the kit of choice. This patent-pending technology reports RNase activity in a convenient and sensitive assay that delivers fast, accurate results in real time. The RNaseAlert Kit uses a novel RNA substrate tagged with a fluorescent reporter molecule (fluor) on one end and a quencher on the other. In the absence of RNases, the physical proximity of the quencher dampens fluorescence from the fluor to extremely low levels. When RNases are present, however, the RNA substrate is cleaved and the fluor and quencher are spatially separated in solution. This causes the fluor to emit a bright green signal (when excited by light of the appropriate wavelength) that can be readily detected by eye (with illumination on a UV box) or with a filter-based or monochromator-based fluorometer. The sequence of the RNaseAlert Substrate has been carefully optimized for sensitive detection of several RNases, including RNase A, RNase T1, RNase I, micrococcal nuclease, S1 nuclease, mung bean nuclease, and Benzonase®.
RNaseAlert is provided in two convenient kit formats. The RNaseAlert Lab Test Kit is suitable for testing small sample numbers and can be used to ensure that solutions, tubes, tips, etc. are RNase-free. The RNaseAlert QC System is designed for high throughput assays in a 96 well format. This kit contains enough substrate to test 480 samples (5 x 96) using a fluorometer.
Common sources of RNase contamination include lab benches, pipettors, glassware, tubes, tips and electrophoresis apparatus. Ambion's RNaseZap® provides an easy and effective way to decontaminate these surfaces. RNaseZap is not simply a detergent that spreads contamination around (like many other commercially available products), but contains three ingredients that are active against RNases. It has proved to be effective at removing contamination from glassware, plastic, countertops, and pipettors. RNaseZap is fast and convenient, destroying RNases immediately on contact. Simply apply the solution to the surface to be treated and rinse with nuclease-free water.
RNaseZap Wipes are a new and unique RNase control product designed for the elimination of surface RNases. RNaseZap Wipes are towelettes pre-soaked in an RNaseZap formulation, which allows RNases to be destroyed with a simple wipe.