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RNase Detection: Tools to Identify Sources of Contamination
To determine if your lab or samples have been contaminated, test for sources of contamination. A common contaminant in molecular biology labs is RNase or ribonuclease. RNases are proteins that target and degrade RNA. There are several types of RNases, and they’re ubiquitous on surfaces, hands, and air. To combat RNase contamination in labs, RNase detection tools are available to monitor and measure for RNase activity.
Detect RNases in any lab—RNaseAlert Lab Test Kit
The RNaseAlert Lab Test Kit is a 25-reaction, microcentrifuge tube-based assay with results read using a UV light source.
The RNase detection kit is designed to detect 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 handheld light). The fluorescence intensity is directly proportional to the amount of RNases present. 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 via fluorometer.
High-throughput RNase detection using RNaseAlert QC System
The RNaseAlert QC System is designed for quantitative high-throughput RNase monitoring using a microplate fluorometer capable of detecting FAM (fluorescein). It comes with enough 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 more reproducible and quantitative measurements.
What is the function of RNase?
RNases are enzymes that catalyze cleavage on RNA, contributing to the degradation of RNA. RNases are important in critical cell and biological processes. However, RNase presence is unwanted in lab spaces where researchers work with RNA. This is one of the reasons why RNA is difficult to work with—RNases are common everywhere and preventing RNase contamination is crucial to protect your experimental results and research. Types of RNases that can be detected include:
- RNase A
- RNase B
- RNase T1
RNase detection to protect experimental results
If working with RNA, RNase contamination is undoubtedly a potential problem because RNases readily degrade RNA. If lab spaces or samples become contaminated with RNases, they can be tested for the presence of RNases by detecting RNase activity.
RNase detection in water sources is recommended at least monthly. For bench-prepared reagents, surfaces, and samples, scientists recommend detecting RNase activity on an as-needed basis.
RNase detection kits
RNase detection kits are available to determine if buffers, reagents, or lab spaces are contaminated with RNase. When choosing an RNase detection kit, it is important to identify what types of RNases the kit detects, as well as the kit’s sensitivity (lowest detection limit of the assay). Most RNase detection kits detect the presence of RNase activity via a fluorescence-based assay.
The RNaseAlert assay: quick and sensitive
The RNaseAlert RNase detection kit is a sensitive and easy to use fluorescence-based assay that can detect as little as 3.5 x 10-7 units (∼0.5 pg) of RNase A in a thirty-minute protocol. The RNaseAlert Kit uses an RNA substrate tagged with a fluorescent reporter on one end and a quencher on the other. In the absence of RNases, the quencher and reporter are near one another. When RNases are present, however, the quencher and reporter are spatially separated in solution as RNA substrates are cleaved. This causes a fluorescence signal that can be readily detected by UV illumination 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: 1) RNaseAlert Lab Test Kit is suitable for testing small sample numbers and can be used to help ensure that solutions, tubes, tips, etc. are RNase-free. 2) 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.
NOTE: Monitor nuclease contamination during routine protein purification—an extremely useful application of the RNaseAlert assay is testing protein fractions for the presence of contaminant RNases during protein purification. Elimination of all traces of RNases during protein purification can sometimes be a daunting task. Simultaneously monitoring the elution profile of both the desired protein and contaminating nucleases aids in the design of a purification scheme which separates RNases from the desired protein. Most column fractions can be monitored directly in the RNaseAlert assay if the ionic strength and pH are not inhibitory to any contaminating RNases. RNases are active in salt solutions below 200 mM in a pH range of 6–9, thus buffers of greater ionic strength can be diluted in water or 1X RNaseAlert buffer prior to testing.
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