RNase contamination is a potential problem for anyone who works with RNA. Researchers at both small academic research laboratories and large biotechnology corporations have similar concerns about RNase contamination. Ambion and Integrated DNA Technologies, Inc. have co-developed the RNaseAlert® Kit, a sensitive and easy to use fluorescence-based assay that can detect even trace amounts of RNase. Now, it is feasible and economical for you to test and re-test critical solutions that come in contact with your precious RNA.
Fast Procedure with Sensitive Results
The RNaseAlert assay is very simple. An optimized RNA oligonucleotide, double-labeled with both fluorescent and quenching moieties, is introduced as a target for any contaminating RNase. In the presence of RNase, the substrate is cleaved, releasing the fluor which then fluoresces. The fluorescent signal can be detected by eye or with a fluorometer (see Figure 1). Results are obtained in less than one hour.
Figure 1. Schematic of RNaseAlert™ Procedure.
The most common method for detecting RNase contamination in solutions is to incubate an RNA substrate with a test solution and then check for degradation of the RNA by ethidium bromide stained agarose gel electrophoresis. This assay has very low sensitivity and requires a subjective judgement. Other more sensitive tests for RNase require a radio-labeled RNA substrate or a fluorescence polarization instrument and are time consuming and costly. The RNaseAlert technology is extremely sensitive and can detect 0.5 pg RNase A or less, much less than most common RNase tests. In addition, the RNaseAlert procedure is neither time-consuming nor requires specialized equipment. The assay is non-toxic and includes all the necessary reagents to perform your own quality control assays. Ambion scientists have successfully used the RNaseAlert kit to detect many types of RNases from various sources. While stable in the absence of RNase, the fluorescence substrate can be cleaved under a wide range of conditions, making this assay useful for examining most molecular biology solutions or surfaces for contaminating RNases.
Detect RNases in Any Lab
The RNaseAlert Kit is available in two formats to accommodate both occasional and high-throughput users. The RNaseAlert Lab Test Kit is a 25-reaction, microcentrifuge tube based assay with results read using a UV light source, whereas the RNaseAlert QC System is designed for use in a microplate fluorometer and contains reagents and plates for 480 (5 x 96) reactions.
The RNaseAlert Lab Test Kit is designed to indicate contaminating RNases in almost any solution by a clear visual read-out. The lyophilized substrate is dissolved in the provided reaction buffer, the sample to be tested is added, and the assay is incubated at 37°C. The results are visualized by irradiating the tube with a UV light source (transilluminator or hand held light) and looking for a bright green fluorescent glow. The fluorescence intensity will be directly proportional to the amount of RNase contamination. This assay is typically completed in less than one hour. For a permanent record of the results, tubes can be photographed using a gel documentation system. If a quantitative value is needed, the fluorescence can be quantified in a fluorometer. For routine or higher throughput RNase monitoring, the RNaseAlert QC System is recommended.
High Throughput RNase Detection
The RNaseAlert QC System is designed for quantitative high throughput RNase monitoring using a microplate fluorometer capable of detecting FAM (fluorescein). It comes with substrate for 480 assays in a 100 µl format (optimized for a 96-well plate). Rather than using a visual assay, the fluorescence generated by RNase cleavage of substrate is quantified in a fluorometer. If desired, the assay can be monitored in real-time by measuring fluorescence every 2-5 minutes over a specific time period (typically 10-60 minutes). This type of assay is very useful for reproducible and quantitative measurements (see Figure 2).
Figure 2. Real-time Fluorescent Monitoring of RNase A Activity.
The indicated amounts of RNase A (0-0.2 pg) were monitored in the RNaseAlert QC System during a 1 hour incubation at 37°C.