EBC New Product Release | See the micro, DNases & RNases
In recent years, the use of mRNA synthesis technology has become more and more extensive, and the requirements for quality control in mRNA vaccines, drug research and production processes have also been increasing. Deoxyribonuclease (DNases) and RNases are enzymes that are ubiquitous in the environment and many biological materials. They can degrade DNA or RNA, influencing many research and production processes involving DNA and RNA. Due to the fragility of mRNA molecules, it is extremely important to inhibit or eliminate nuclease activity in research and production environments.
There are two main problems in the detection of DNases and RNases residues. On the one hand, since the content of DNases and RNases residues in the research and development, production environment and raw materials is in an unknown state, the methods for detecting their existence must be sensitive enough; On the other hand, the timeliness of testing must also be taken into account to ensure that the relevant R&D and production progress will not be affected.
To this end, we need a fast and sensitive method to detect the nuclease activity in the environment (such as experimental tables, pipette tips, reservoirs, reactors and other consumables) and materials (such as antibodies, enzymes, buffers), determine whether there is DNases or RNases pollution, and be able to quantitatively assess the degree of contamination, so as to develop a corresponding removal plan to meet the needs of R&D and production conditions.
The method of defining pollution-free is not clearly defined in documents such as USP43, EP10 and Chinese Pharmacopoeia 2020 Edition. The quorum limit of nucleic acid hydrolysis-UV spectrophotometry is only 0.01U/μL, which is not suitable for the detection of activity of trace nucleases. The nucleic acid hydrolysis-gel electrophoresis method is greatly affected by the subjective judgment of the experimenter and cannot be accurately quantified, and the long operation time also reduces the measurement flux. High performance liquid chromatography (HPLC) and electrochemical methods are time-consuming, laborious, and limited by instrumentation.
In this regard, some researchers [1] proposed the fluorescent probe method, which not only has high sensitivity and fast detection speed, but also can realize the quantitative detection of nuclease activity. The DNase/RNase fluorescent probe detection kit is one of the best choices.
After repeated exploration and continuous optimization, Hzymes Biotech launched the HS-DNase Detection Kit (fluorescent probe method) and the HS-RNase detection kit (fluorescent probe method) to fully meet the high sensitivity and timeliness problems in DNases and RNases detection.
This high-sensitivity detection kit is the first time launched in China!
Detection principle:
The principle of DNase and RNase detection kits is the same. DNA probes and RNA probes labeled with fluorophores are designed and mixed with the test sample.When the sample does not contain DNases or RNases activity, the probe is stable, the fluorophore and the quenching group are closer together, and no fluorescence signal is generated due to the principle of fluorescence resonance energy transfer; when the sample contains DNases or RNases activity, the probe is degraded and the fluorophores and quenching groups are moved away from each other, resulting in a gradually enhanced fluorescent signal; The rate of increased fluorescence is positively correlated with the number and activity of enzymes.
The principle is shown in the following figure:
Schematic diagram of the principle of fluorescent probe method
Schematic diagram of the reaction curve of the DNase/RNase detection kit
Scope of application:
It is used to detect DNase contamination of the environment, materials, etc. in scientific research and production processes
HS-DNase Detection Kit (Fluorescent Probe Method)
Features:
High sensitivity: detection limits as low as 1.25× 10-6 U/μL (DNase I), which is 1/8 of the existing detection method
High timeliness: The test results are obtained immediately after 30 minutes of reaction.
- Sensitivity is superior to gel electrophoresis
The HS-DNase assay kit is compared to nucleic acid hydrolysis-gel electrophoresis to determine the same low-concentration enzyme sample as shown.
HS-DNase detection kit measures 3×10-5U/μL DNase I, which can be clearly distinguished from 0 concentration
Electrophoresis for the determination of DNase I, 9/10 channel (3×10-5 U/μL) and 1 to 8 channel (≤ 3×10-6 U/μL), 17 channel negative control can not be clearly distinguished.
- The sensitivity is superior to that of similar fluorescent probe methods
Compared with the same type of method (fluorescent probe method), the lower detection limit of the HS-DNase detection kit is as low as 1/8
When the sample concentration is 6.25×10-6U/μL, the DNase detection kit of the same type of method is indistinguishable from the negative control.
When the sample concentration is 6.25×10-6U/μL, the HS-DNase detection kit can be clearly distinguished from the negative control
When the concentration of DNase I is low (1×10-6~10×10-6U/μL), the S/N value measured by the DNase detection kit of the same type is close to 1, and the HS-Dnase detection kit is multiplied.
The main components of the HS-DNase detection kit are:
HS-DNase Detection Kit Components
Note: The standard is DNase I, and its active unit is defined as the amount of enzyme required to completely degrade 1 μg of pBR322 DNA within 10 min at 37 °C in DNase I reaction buffer[1], and one DNase I active unit is equivalent to 0.3 Kunitz units[2].
Q&A
- What is the reaction temperature and time of the HS-DNase assay?
Please place the reaction system under 37 °C constant temperature conditions for experiments, the reaction time is 30 minutes.
- Under what circumstances can false positive or false negative results or quantitative results be inaccurate?
- Gel buffers, high concentrations of viscous substances, surfactants and dark solutions may interfere with fluorophorephorec luminescence;
- If the sample solution to be measured contains substances that inhibit the activity of DNases, the determination result is the overall enzyme activity of the sample solution, not the activity of the enzymes in it. These substances include:
- High ionic strength solutions (e.g. 5M NaCl, 20x SSC, 3M sodium acetate, etc.).
- Buffer with pH<4 or pH >9
- Defusions, detergents, chelating agents, or any solution that denatures proteins (e.g., SDS, guanidine thiocyanate, urea, EDTA, etc.).
- Solutions that cause instability in the chemical properties of DNA probes, such as pH>9 solutions, caustic solutions (strong acids and strong bases, bleaching agents, etc.).
- When preparing a standard solution, why should the previous steps dilute with a standard dilution instead of with DNase-free RNase water?
Because the standard is relatively stable in the standard dilution, even multiple dilutions will not change its own activity. If diluted with water, multi-step operation may cause the activity of the standard to change, and the standard curve will be biased.
- How do I adjust the appropriate microplate reader gain value?
If your microplate reader has an auto gain option, use auto gain. If your microplate reader does not have an automatic gain option, set the middle value according to the gain value range, and then adjust the appropriate gain value according to the fluorescence value of the positive control well after the reaction (79 μL DNase-free RNase water + 1 μL DNase I standard): if the instrument upper limit is exceeded, the gain value is appropriately lowered, and if it is much lower than the instrument upper limit, the gain value is appropriately adjusted.
- Why are severely contaminated samples likely to have false-negative results?
The criteria for determining DNase contamination are: RFU30 (sample to be measured) ≥2× RFU0 (sample to be measured), that is, the RFU value at 30 minutes of reaction reaches more than 2 times the RFU value of 0 minutes. If the sample is heavily contaminated, it is possible that the reaction will start quickly, with a very high RFU0 value measured in a very short period of time, resulting in a false-negative result of RFU30 (sample to be measured) <2× RFU0 (sample to be tested). At this point, the sample to be tested needs to be diluted with DNase-free RNase-free water.
- If the RFU value of the negative control is not 0, does the negative control be contaminated?
Not necessarily. Negative controls are usually able to measure lower RFU values, but do not increase significantly with the lengthening of reaction time, and it is generally believed that 30 min RFU30 is less than 0 min RFU0 twice that the negative control is not contaminated.
Product Information:
Catalog No | Product Name | Test Data |
HBP002902 | DNase Assay Kit(Flourscence Prob Method) | 192 Test |
HBP002903 | DNase Assay Kit(Flourscence Prob Method) | 48 Test |
RNase Detection Kit (Fluorescent Probe Method)
Scope of application: It is used to detect RNase contamination of the environment, materials, etc. in scientific research and production processes.
Features:
High sensitivity: detection limits as low as RNase A: 0.313 pg/mL, approximately 1.56×10-9U/μL, 1/8 of the same type of method assay kit
High timeliness: The test results are obtained immediately after 30 minutes of reaction
Sensitivity is superior to gel electrophoresis
The HS-RNase assay is compared to nucleic acid hydrolysis-gel electrophoresis to determine the same low-concentration enzyme sample, as shown.
HS-RNase detection kit detects RNase A, 2.5×10-8U/μL and 0 concentration can be clearly distinguished.
Electrophoresis assay for RNase A, channels 1 to 8 (5×10-8U/μL) were indistinguishable from negative controls of channels 17, 18 (2.5×10-8U/μL) and 19.
The sensitivity is better than that of the same type of detection method
Compared to the same type of detection method (fluorescent probe method), the lower detection limit of the HS-RNase assay kit is as low as 1/8
When the sample concentration is 3.13×10-9U/μL, the RNase detection kit of the same type is indistinguishable from the negative control.
When the sample concentration is 3.13×10-9U/μL, the HS-RNase detection kit can be clearly distinguished from the negative control
At low concentrations of RNase A (1×10-6~10×10-6U/μL), the S/N value detected by the RNase detection kit of the same type of detection method is between 1 and 2, and the HS-RNase detection kit is multiplied.
HS-RNase assay kit main components:
Q&A
- What is the reaction temperature and time of the HH RNase assay?
Please place the reaction system under 37 °C constant temperature conditions for experiments, the reaction time is 30 minutes.
- Under what circumstances can false positive or false negative results or quantitative results be inaccurate?
- The presence of substances that may interfere with the luminescence of the fluorophore, which may lead to false-negative results;
- If the sample solution to be measured contains substances that inhibit RNAse activity, it may lead to false negative resultsc. Solutions that cause instability in the chemical properties of RNA probes, which may lead to false positive or false negative results
- When preparing a standard solution, why should the previous steps dilute with a standard dilution instead of with DNase-free RNase water?
Because the standard is relatively stable in the standard dilution, even multiple dilutions will not change its own activity. If diluted with water, multi-step operation may cause the activity of the standard to change, and the standard curve will be biased.
- How do I adjust the appropriate microplate reader gain value?
If your microplate reader has an auto gain option, use auto gain. If your microplate reader does not have an automatic gain option, set the mid value according to the gain range, and then adjust the appropriate gain value according to the fluorescence value of the positive control well after the reaction (79 μL DNase-free RNase water + 1 μL RNase A standard): if the instrument upper limit is exceeded, the gain value is appropriately lowered, and if it is much lower than the instrument upper limit, the gain value is appropriately increased.
- Why are severely contaminated samples likely to have false-negative results?
The criteria for determining RNase contamination are: RFU30 (sample to be measured) ≥2× RFU0 (sample to be measured), that is, the RFU value at 30 minutes of reaction reaches more than 2 times the RFU value of 0 minutes. If the sample is heavily contaminated, it is possible that the reaction will start quickly, with a very high RFU0 value measured in a very short period of time, resulting in a false-negative result of RFU30 (sample to be measured) <2× RFU0 (sample to be tested). At this point, the sample to be tested needs to be diluted with DNase-free RNase-free water.
- If the RFU value of the negative control is not 0, does the negative control be contaminated?
Not necessarily. Negative controls are usually able to measure lower RFU values, but do not increase significantly with the increase in reaction time, and it is generally believed that 30 min RFU30 is less than 2 times of RFU0 min, indicating that the negative control is not contaminated.
Product information
Catalog No | Product Name | Test Data |
HBP003002 | RNase Assay Kit(Flourscence Prob Method) | 192 Test |
HBP003003 | RNase Assay Kit(Flourscence Prob Method) | 48 Test |
References
[1] New England Biolabs DNase I
[2] Kunitz M. Crystalline Desoxyribo – nuclease I. Isolation and General Pro – perties Spectrophotometric Method for the Measurement of Desoxyribonuclease Activity[J]. The Journal of General Physiology, 1950, 33(4):349-362.
[3] Thermo scientific RNase A(DNase and Protease-free)
[4] Kunitz, M. A. A spectrophotometric method for the measurement of ribonuclease activity[J]. Journal of Biological Chemistry, 1946, 3(2):308-320.