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Cat. No.: RAFA-100 (for 100T)

Cat. No.: RAFA-500 (for 500T)

Description

RNase Activity Fluorometric Assay Kit, or Fluorometric RNase Assay Kit, is a kit designed for the rapid and highly sensitive detection of ribonuclease (RNase) activity, such as RNase A, using a fluorometric method.

Ribonucleases, similar to deoxyribonucleases (DNases), are widely present in laboratory environments and biological samples. Since nucleases can degrade nucleic acids, their presence can interfere with many experiments. Traditional methods for detecting RNase activity are often time-consuming and have relatively low sensitivity.

This kit offers high sensitivity, capable of detecting as little as 0.3 pg of RNase A. It is also known as the Ribonuclease Activity Fluorometric Assay Kit, RNAse Activity Fluorometric Assay Kit, RNase A Activity Fluorometric Assay Kit, Fluorometric RNase Activity Assay Kit, or Fluorometric RNase A Activity Assay Kit.

Ribonucleases (RNases) are enzymes that catalyze the degradation of RNA by hydrolyzing the phosphodiester bonds of RNA. They are classified into endoribonucleases and exoribonucleases. The most common RNases include RNase A, RNase T1, and RNase H. RNase A and RNase T1 are commonly used to hydrolyze RNA in DNA or protein samples, both showing high activity towards single-stranded RNA. RNase H specifically hydrolyzes RNA in DNA-RNA hybrids but does not hydrolyze the phosphodiester bonds in single-stranded or double-stranded DNA or RNA.

The RNase Activity Fluorometric Assay Kit uses the Fluorescence Resonance Energy Transfer (FRET) method. The principle of detection is illustrated in Figure 1. The RNase substrate is a synthetic RNA oligonucleotide probe with a FAM fluorophore (donor) at one end and a TAMRA quencher (acceptor) at the other end. These two groups have overlapping absorption spectra. When the distance between the two fluorophores is appropriate, the fluorescence energy is transferred from the donor to the acceptor, causing a decrease in the fluorescence intensity of the donor. When the substrate is cleaved by RNase, the ends of the substrate separate, and the two groups are no longer in proximity, resulting in the detection of FAM fluorescence. This allows for highly sensitive detection of nuclease activity. The maximum excitation wavelength of FAM is 490 nm, and the maximum emission wavelength is 520 nm.

Components

• 10X Reaction Buffer
• RNase A (10mg/ml)
• RNase Substrate
• Nuclease-free Water

Features

• This kit offers high sensitivity and requires only a small amount of sample. It includes an RNase A positive control to facilitate the establishment of the detection system. The RNase substrate probe has been optimized for high sensitivity, capable of detecting as little as 0.3 pg of RNase A activity. RNase A shows a good linear relationship in the range of 0-10 pg, allowing for the calculation of RNase A activity in samples by setting up a standard curve.
• This kit has a wide range of applications, is flexible to use, and provides fast detection. It can be used not only for the quantitative detection of RNase A but also for various other nucleases. Compared to the ELISA method, it is simpler, faster, and more accurate. Experimental validation has shown that this kit can be used to detect the relative activity of enzymes such as Mung Bean Nuclease and S1 Nuclease. However, since RNase H specifically hydrolyzes RNA in DNA-RNA hybrids, this kit is not suitable for detecting RNase H. It is also not very suitable for the high-sensitivity detection of RNase T1 and Broadonase or Benzonase activity. The kit uses a one-step detection method, which is simple and fast, taking only about 20 minutes to complete.

Storage

Store at -20ºC, valid for one year. The RNase Substrate should be stored protected from light.

Precautions

• Since ribonucleases are widely present in the environment and relatively stable, it is recommended to perform RNase activity detection in a relatively clean environment, such as a laminar flow hood or biosafety cabinet, to avoid contamination of the samples by environmental RNases.
• The 10X Reaction Buffer, RNase Substrate, and Nuclease-free Water should be completely thawed and equilibrated to room temperature before use, as failure to do so may affect the detection results. RNase A (10 mg/ml) should be kept on ice during use, and all reagents should be stored under the conditions specified by the kit immediately after use.
• Ensure that the sample pH is between 7-8, or that the pH of the reaction system is between 7-8 after adding the sample, as deviations may affect the signal value and stability of the detection results. For small volume reagents, it is recommended to centrifuge briefly to collect the liquid at the bottom of the tube before use. Frozen reagents must be completely thawed and mixed thoroughly before use.
• This kit may not be suitable for some ribonucleases, such as RNase H. Generally, the 10X Reaction Buffer provided in this kit is universal for most nucleases, but there may be exceptions for certain specific enzymes. If necessary, use the specific nuclease buffer to dilute and react with the sample.
• Take care to prevent contamination of reagents by RNase during operation.
• For detection, it is recommended to use a 96-well black plate, preferably 96-Well Black Opaque Plates.
• This product is for scientific research use only by professionals and is not intended for clinical diagnosis or treatment, food, or drugs, and should not be stored in a residential environment.
• For your safety and health, please wear a lab coat and disposable gloves when handling.

Related: DNase Activity Fluorometric Assay Kit

Only for research and not intended for treatment of humans or animals

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