In recent years, studies have shown that mRNA will occur specific degradation if dsRNA, formed by sense RNA and antisense RNA, is transferred into cell, and eventually it leads to target genes silence. The post-transcriptional gene silencing (PTGS) is described as RNAi. Molecular mechanisms of RNAi
RNAi consists of inititation and effector steps. At inititation steps, the dsRNAs get processed into 21-23 nucleotide (nt) small interfering RNAs (siRNAs) by an RNase III-like enzyme called Dicer. And the 3 'end of each siRNAs fragment has two bases prominent. At effector steps, the siRNAs assemble into endoribonuclease-containing complexes known as RNA-induced silencing complexes (RISCs), unwinding in the process. The siRNA strands subsequently guide the RISCs to complementary mRNA molecules, where they cleave and destroy the cognate RNA. In plants, dsRNA that contain promoters are also cleaved into 21-23 nucleotide (nt), and transcriptional gene will silence by methylation of homologous DNA sequences.
Design of siRNA
1. Firstly, screen target sequences of RNAi. Then start with the start codon “AUG” of mRNA, look for the sequence “AA”, and write down 19 base sequences of the 3 '-UTR as potential siRNA target sites. Studies have shown that siRNA with an about 45% - 55% GC content is more effective than those with higher GC levels.
2. Compare the potential sequences with the corresponding genetics databases, exclude sequences which have a significant homology with EST and other coding sequence. (e.g. using BLAST( www.ncbi.nlm.nih.gov/BLAST/)).
3. Select the appropriate target sequence for synthesis. In order to find the most effective siRNA sequences, the gene of interest usually needs to design several target sequences.
4. Set negative control group. The siRNA of negative control should have the same composition, but it does not share any significant homology with mRNA. A common approach is to upset the selected sequence of siRNA. The results should also be checked to ensure that it does not have the homology with other genes in the target cell. Preparation of siRNA
So far, the common methods of preparation of siRNA include chemical synthesis, in vitro transcription, digestion of long fragment dsRNA with RNase III, and expression of siRNA through the RNAi expression vector, virus vector or siRNA expression cassettes in cells.
1. Chemical synthesis
Chemical synthesis is applicable when the most effective siRNA has been found and a large amount of siRNA studies need be conducted. While it is expensive and the method has a long custom cycle, especially with special needs.
2. In vitro transcription
DNA Oligo is used as template, and siRNAs are synthesized by in vitro transcription. Compared with chemical synthesis, the cost is lower and it is faster to obtain siRNA. Moreover siRNA also has less toxicity, better stability and higher efficiency of transfection than chemical synthesis. But the scale of the experiment is limited, in vitro transcription is unsuitable for extensive research.
3. Preparation of siRNA by digestion of long fragment dsRNA with RNase III
When 200-1000 nucleotide (nt) of target mRNA are used as template, prepare long fragment dsRNA by in vitro transcription, then digest dsRNA with RNase III (or Dicer) in vitro. After removing the dsRNA that has not been digested, transduce directly into cells. The main advantages are that it reduces the steps of detecting and screening effective siRNA sequences, it can save time and money for researchers, while it may cause nonspecific gene silencing.
4. SiRNA expression vector
Most siRNA expression vectors need rely on one of promoters, RNA polymerase III type 3 promoter, to initiate the expression of gene sequence which encode small hairpin RNA (shRNA) in mammalian cells. These promoters include human U6 promoter, mouse U6 promoter, and human H1 promoter. The siRNA expression vector with antibiotic markers can continuously inhibit the expression of target genes in cells, is suitable for long-term study. Virus vectors can also be used for the expression of siRNA. It can make specific gene silence with high efficiency by directly infecting cells, and the transfection rate is much higher.
5. SiRNA expression cassettes
SiRNA expression cassettes (SECs) is a siRNA expression template by PCR amplification. It includes the RNA promoter pol III, a hairpin structure of siRNA, and the RNA termination site pol III. SECs can be introduced directly into cells without cloning and sequencing. It spends shorter time. So SECs is the most effective tool for screening siRNA. If the suitable restriction sites are added by PCR, a siRNA expression vector can be constructed through cloning after selecting the most effective siRNA. And the vectors can be used to express stably siRNA. SiRNA transfection
There are five main methods for introducing siRNA expression vector and siRNA expression cassettes into eukaryotic cells, including calcium phosphate precipitation, electroporation, DEAE-dextran or polybrene, mechanical method, and cationic liposome reagent. In order to achieve high transfection efficiency, pay attention to the following things during transfection experiments:
1. Purify siRNA;
2. Avoid contamination of RNase;
3. Ensure repeatability of transfection by using healthy cells and strict operation;
4. Avoid the use of antibiotics;
5. Select the appropriate transfection reagent;
6. Optimize conditions of transfection and detection through the appropriate positive control;
7. Optimize experiment by using siRNA labeling.