| Product Description | Aladdin's RNase III is a dsDNA-specific ribonuclease derived from Escherichia coli, expressed and purified using the PerfectProtein™ Technology Platform developed by aladdin. This product specifically cleaves double-stranded RNA (dsRNA) in the presence of manganese (Mn2+), producing 18-25bp interference RNA (siRNA) with 2-3 protruding bases at the 3' hydroxyl end [1,2]. Similar to the products generated by Dicer enzyme, the siRNA has various applications such as RNA interference, gene silencing, and target validation in mammalian cells [3]. RNase III cannot hydrolyze DNA or single-stranded RNA.Please refer to Figure 1 for the cleavage of dsRNA (500bp) by this product to generate siRNA.Figure 1. Generation of siRNA from dsRNA (500bp) using Aladdin's RNase III . In a 10µl reaction (50mM Tris-HCl, 50mM NaCl, 1mM DTT, 20µM MnCl2 (pH7.5 at 25℃)), add 1µg of dsRNA (500bp) and a specified amount of this product or N Company's (Competitor) RNase III as indicated in the figure. The reaction was incubated at 37℃ for 20 minutes, then terminated by adding EDTA. The reaction product was mixed with 6X DNA Loading Buffer , and subjected to 1% agarose gel electrophoresis. The experimental results were observed under UV light. As shown in the figure, this product exhibited similar performance to N Company's RNase III. The dsRNA was generated by in vitro transcription of dsDNA containing a T7 promoter using the T7 Quick High Yield Transcription Kit . The resulting complementary 500nt single-stranded RNAs were annealed using the Annealing Buffer for RNA Oligos (5X) following the product manual. This figure is for reference only, which may vary depending on different experimental conditions.s
Application： Hydrolysis of dsRNA; production of siRNA; gene silencing; target validation.
Source： Purified from E. coli with expression of recombinant RNase III.
Enzyme storage buffer： 10mM Tris-HCl, 500mM NaCl, 1mM DTT, 0.5mM EDTA, 50% Glycerol (pH8.0 at 25℃).
Inactivation or inhibition： This enzyme can be inactivated by EDTA at a final concentration of 50mM. Heat inactivation of this enzyme can not be performed.
Precautions： The reaction can be terminated by adding EDTA to a final concentration of 50mM. Do not perform heat inactivation of RNase III. Otherwise, the yield of siRNA will be reduced.RNA is highly susceptible to degradation. Please handle RNA samples with extreme care to avoid RNase contamination.RNase III should be kept on ice during use, and should be stored at -20°C immediately after use.This product is for R&D only. Not for drug, household, or other uses.For your safety and health, please wear a lab coat and disposable gloves during the operation.
Instructions for Use： 1. Preparation of siRNAa. Set up the reaction (total volume of 20 or 100μl) on ice as follows:ReagentVolumeVolumeFinal ConcentrationdsRNAxμl (2μg)xμl (10μg)100ng/µl10X Reaction Buffer2μl10μl1X10X MnCl22μl10μl1XNuclease-Free Water(14-x)μl(70-x)μl-RNase III2μl10μl0.2U/µlTotal Volume20μl100μl-Note 1: To achieve the best digestion effect, the enzyme dosage can be adjusted as appropriate. Note 2: If multiple reactions are carried out simultaneously, prepare a mastermix of all components in the table except for RNase III. Aliquot the mastermix into each reaction tube, then add RNase III at the end. b. Mix the reaction well gently and centrifuge briefly to collect liquid to the bottom. c. Incubate at 37℃ for 20 minutes. Note: The reaction time can be adjusted appropriately as required. d. Terminate the reaction by adding 10X EDTA to inactivate RNase III. Note: Heat inactivation should not be performed as it reduces the yield of siRNA.2. For other applications, please refer to relevant literature.References:1. Morlighem JE, Petit C, Tzertzinis G. Biotechniques. 2007. 42(5):599-600, 602, 604-6.2. Court DL, Gan J, Liang YH, Shaw GX, Tropea JE, et al. Annu Rev Genet. 2013. 47:405-31.3. Donzé O, Picard D. Nucleic Acids Res. 2002. 30(10):e46.
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