Comparison of 5 Methods and Pathways for Synthesizing siRNA Molecules-RNAi

At present, researchers use the method of introducing siRNA molecules into cells for RNAi research. There are currently 5 methods and pathways for synthesizing siRNA molecules:

1. Chemical synthesis;

2. In vitro transcription;

3. "Cocktail" method (RNaseIII enzyme digestion of dsRNA);

4. In vivo expression of siRNA mediated by plasmids and viral vectors;

5. siRNA expression box mediated siRNA in vivo expression.

The first three methods involve obtaining siRNA molecules in vitro and directly introducing siRNA into mammalian cells using methods such as transfection and electroporation; The latter two methods use vectors and siRNA expression frames as mediators to express siRNA molecules in cells. Experimenters can choose specific methods for conducting experiments based on the purpose and situation of the experiment.

Except for the RNaseIII enzyme digestion method for dsRNA, specific siRNA sequences must be designed based on the target gene before synthesizing siRNA using the other four methods mentioned above. To obtain a highly efficient siRNA, it is generally necessary to design 3-4 siRNAs and conduct experiments and screening based on them.

In order to help improve the efficiency of designing siRNA, Cenix BioScience, a partner of Ambion, has developed software specifically for designing siRNA, which can greatly reduce the number of siRNA tests and enable researchers to quickly obtain highly efficient siRNA. (Details: www.ambion. com/siRNA)

After the customer designs the siRNA sequence, the synthesizer synthesizes and purifies it according to the sequence. Ambion can provide synthesis services based on customers' siRNA sequences, or use Cenix BioScience's professional software to design sequences for customers before synthesis.

The siRNA products synthesized by Ambion have been tested by MALDI-TOF and relevant data has been provided. Its purity is divided into three categories: ordinary grade (desalination, deprotection, purity greater than 80%), HPLC grade (purity greater than 97%), and PAGE grade (purity greater than 97%).

The advantage of this method is that the obtained siRNA has high purity and does not require any other experiments, but the disadvantage is that it is expensive. So currently, researchers mostly use relatively inexpensive methods to synthesize siRNA for screening, and after confirming high-efficiency siRNA, use chemical synthesis to synthesize siRNA for experiments. In vitro transcription is an inexpensive method.

The corresponding DNA strand is synthesized for siRNA sequence, and then transcribed in vitro using RNA polymerase. The transcription product can be introduced into cells. Ambion's SilencerTM siRNA Construction Kit is a kit that uses this method.

The advantage of this method is that it is inexpensive and the reagent kit is easy to use, but the disadvantage is that it requires other experiments and the synthesis amount of siRNA is limited.

Selecting the most efficient siRNA molecule from some siRNA sequences is a major bottleneck in RNAi research. In response to this situation, researchers have proposed a siRNA preparation method called "cocktail". This method imitates the working principle of siRNA in vivo, and the process is as follows: firstly, mRNA targeting the target gene is transcribed and synthesized into long dsRNA in vitro; Then use RNaseIII or Dicer to enzymatically hydrolyze the long dsRNA into a mixture of siRNAs; Finally, introduce the mixture into the cells. Ambion's SilencerTM siRNA Cocktail Kit is a kit that uses this method.

The advantage of this method is that it can avoid tedious siRNA design and screening work, but the disadvantage is that it may produce non-specific gene inhibition, and after effectively inhibiting gene expression, the truly effective siRNA is not known.

The above three methods all involve obtaining siRNA in vitro and then introducing it into cells, but this method has two drawbacks that cannot be overcome: siRNA is easily degraded after entering cells; The amount of siRNA entering the cell is not controlled. In response to this situation, siRNA expression mediated by plasmids and viral vectors has emerged in vivo. The basic idea of this method is to clone the DNA double stranded sequence corresponding to siRNA into a vector, located after the promoter of RNA polymerase III, so that the desired siRNA molecule can be expressed in vivo.

The advantage of this method is that it allows siRNA to be directly expressed in vivo, making it the only method used for long-term RNAi research. Resistance markers on vectors can help quickly screen for positive clones. Currently, some companies, including Ambion, have developed viral vectors (such as adenovirus vectors) that allow siRNA to enter more types of cells for research. Due to the involvement of clone construction in this method, extensive experiments such as clone construction and sequence determination are required. Now Ambion can provide 10 plasmid vectors with different promoters and resistance markers, such as mouse U6 promoter, human U6 promoter, and human H1 promoter. Resistance markers include puromycin, neomycin, and hygromycin.

The essence of an siRNA expression frame is a PCR product containing siRNA molecules that can be cloned into a vector to suppress the expression of specific genes in cells. There is a promoter of RNA polymerase III upstream of the expression box and a termination sequence of RNA polymerase III downstream. SiRNA expression mediated by siRNA expression frame is also a method of in vivo expression. Compared with siRNA expression vector, its advantage is that it can be directly introduced into cells without cloning. However, by adding enzyme cleavage adapters at both ends of the expression frame, it can also be cloned into the vector, which is convenient for customers to use. The SilencerTM siRNA Expression Cassette Kit from Ambion is a kit that uses this method.