14. An RNase Residue Detection Test Kit

Product Information

• Product Name: RNase Residue Detection Test Kit
• Product Number: G3058-100T
• Specification: 100 tests

Product Description

Ribonuclease (RNase) is an RNA-hydrolyzing enzyme that is widely present in the environment and can be found in high concentrations in some biological materials, enzymes, and reagents. This can have a detrimental impact on RNA-related experimental procedures. Therefore, it is essential to detect the presence of RNase residue in any solution or biological material that comes into contact with RNA.

This test kit uses a unique RNA substrate that can rapidly and sensitively detect the activity of RNase. One end of the RNA substrate has a fluorescent group, while the other end has a quenching group. When RNase is absent, the quenching group is close to the fluorescent group, suppressing the fluorescence to a very low level. However, when RNase is present, the RNA substrate is hydrolyzed, separating the fluorescent group from the quenching group, resulting in a significant increase in fluorescence. As the fluorescence of the RNA substrate increases with increasing RNase activity, real-time analysis of RNase and DNase in a single sample can be performed using a fluorescence reader.

This kit provides RNase A as a positive control for the detection system and can detect RNase A at levels as low as 0.5 pg. In addition to detecting RNase A activity, this kit can also detect the activity of enzymes such as RNase T1, Micrococcal Nuclease, S1 Nuclease, Mung Bean Nuclease, and Benzonase. It is also compatible with DNase detection systems. Since RNA substrates and DNA substrates have different fluorescent labels, both substrates can be added to the same reaction system, allowing real-time analysis of RNase and DNase in a single sample.

Storage and Transportation

Transport on wet ice and store at -20°C. Shelf life is 12 months.

Components

• Component Number: G3058-100T
• Components:
  • 10× Reaction Buffer: 2×1 mL
  • RNase A (10 mg/mL): 10 μL
  • 5× RNase Substrate: 200 μL
  • ZnSO4 (100 mM): 200 μL
  • Nuclease-free Water: 20 mL
• User Manual: 1 copy

Preparation Before Use

1. 96-well black enzyme-linked immunosorbent assay (ELISA) plate (Nuclease-free).
2. Nuclease-free pipettes and tips.

Operating Procedure

1. Preparation before configuring the reaction system:a. Dilute a series of RNase A concentrations to create a standard curve: Dilute RNase A (10 mg/mL) with Nuclease-free Water to achieve concentrations of 0.25 pg/µL, 0.5 pg/µL, 1 pg/µL, 2 pg/µL. Include a negative control with no RNase A. Add 10 µL of each dilution to the 96-well ELISA plate, resulting in final RNase A amounts of 0, 2.5 pg, 5 pg, 10 pg, 20 pg.b. Sample preparation: If the sample to be tested is a liquid, it can be used directly. If the sample is a solid, immerse it in Nuclease-free Water to prepare a liquid sample.
2. Prepare the reaction system as shown in the table below:When preparing the reaction system, add the corresponding components of the test kit to the 96-well plate.
   

   Component	Minus-RNase Control	Plus-RNase Control	Experimental Samples
   10× Reaction Buffer	10 μL	10 μL	10 μL
   RNase A	0	10 μL	0
   Sample	0	0	10 μL
   RNase Substrate	2 μL	2 μL	2 μL
   Nuclease-free Water	To 100 μL	To 100 μL	To 100 μL

   If the nuclease being detected is Zn2+-dependent, such as Mung Bean Nuclease or S1 Nuclease, add an additional 1 μL of 100 mM ZnSO4 to the reaction system.

3. Detection: Mix the reaction system by shaking or gently tapping to ensure thorough mixing. Immediately use an ELISA reader for detection, setting the detection temperature to 37°C (if the reader doesn’t have temperature settings, room temperature detection is possible, but enzyme activity may be lower). The excitation/emission wavelengths are 492/518 nm. Continuous detection for 30 minutes with a 2-minute interval (detection time can be adjusted based on RNase content, extending the detection time to 60 minutes with a 5-minute interval for lower content).
4. Calculate the residual amount of RNase A in the sample based on the RNase A standard curve.

Notes

1. Read the product manual carefully before operation.
2. Due to the widespread presence of RNase in the environment, it is essential to work in a laminar flow cabinet or biological safety cabinet to avoid contamination of the test samples.
3. The reaction system should be prepared on ice to prevent premature degradation of RNase Substrate.
4. To ensure accuracy, it is recommended to prepare at least two replicates.
5. If you are not testing for the presence of RNase A in your samples, you can skip the RNase A standard curve preparation.
6. The reaction liquid preparation should be done on ice throughout the process.
7. Protect RNase Substrate from light, and avoid exposure to strong light during reaction liquid preparation.
8. Use Nuclease-free tips and minimize cross-contamination between samples during liquid preparation and distribution.