How to ensure the accuracy of siRNA synthesis?

SiRNA synthesis is an important biotechnology method used to study gene function and RNA interference. To ensure the accuracy of siRNA synthesis, a series of measures and steps need to be taken to ensure that the synthesized siRNA can play the expected role in the experiment. Here are some key steps and techniques to ensure the accuracy of siRNA synthesis:

1. Design appropriate siRNA sequences: The design of siRNA is a step to ensure accuracy. When designing siRNA sequences, it is necessary to avoid homology with non target gene sequences while ensuring that the siRNA matches specific regions of the target mRNA. Usually, software based on RNAi design principles is used to design siRNA sequences, such as siDirect, siRNA Target Finder, etc.

2. Choose the appropriate synthesis method: siRNA synthesis can be carried out through chemical synthesis or genomic synthesis. Chemical synthesis is a fast and efficient method for synthesizing siRNA sequences by linking nucleoside nucleotide units synthesized in anhydrous DMF. Gene combination is a method of synthesizing siRNA through PCR amplification. Suitable synthesis methods need to be selected based on experimental requirements and expected applications.

3. Purity testing: The purity of synthesized siRNA is crucial for its accuracy and effectiveness in experiments. The purity of siRNA is usually tested by polyacrylamide gel electrophoresis or high performance liquid chromatography (HPLC). Ensuring the purity of siRNA is above 90% is an important step in ensuring experimental accuracy.

4. Sequence validation: Before conducting experiments using siRNA, it is necessary to validate the synthesized siRNA sequence. The siRNA sequence can be validated through sequencing technology to ensure consistency between the siRNA sequence and design, and to avoid sequence mismatches or mutations. Sequence validation is a crucial step in ensuring the accuracy of siRNA synthesis.

5. Endogenous control: Using endogenous control in experiments is an important method to ensure the accuracy of siRNA synthesis. Endogenous control is a type of siRNA sequence that is not related to the siRNA target and is used to test the effect of siRNA on non-specific effects. By comparing the experimental results of the control group, the specificity and accuracy of siRNA towards the target gene can be evaluated.

6. Optimize siRNA transfection conditions: The transfection conditions of siRNA also have a significant impact on its accuracy and efficacy. When conducting experiments using siRNA, it is necessary to optimize transfection conditions, including cell density, transfection agent concentration, transfection time, and other parameters, to ensure efficient transfection and efficacy of siRNA.

7. Experimental verification: Experimental verification is the final step to ensure the accuracy of siRNA synthesis. When conducting experiments using siRNA, it is necessary to conduct appropriate control group experiments to evaluate the silencing effect and mechanism of siRNA on target genes. Through experimental verification, the accuracy and effectiveness of siRNA in the experiment can be determined.

Through the above series of steps and techniques, the accuracy of siRNA synthesis can be ensured, and expected research results can be achieved in experiments. The accuracy of siRNA synthesis is crucial for gene function research and RNA interference experiments, and also provides important tools and methods for life science research.