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RNAi Protocol
RNA interference (RNAi) is an evolutionarily conserved defense mechanism against transgenic or foreign viruses. RNA interference (RNAi) by double stranded RNA (dsRNAs) molecules of approximately 20-25 nucleotides termed short interfering (siRNAs) is a powerful method for preventing the expression of a particular gene. This process belongs to the mechanism of post-transcriptional gene silencing.
Molecular mechanisms of RNAi
RNAi occurs by a two-stage mechanism: initiation and effector stages. At inititation steps, the dsRNAs get processed into 21-23 nucleotide (nt) small interfering RNAs (siRNAs) by an RNase III enzyme called Dicer. And the 3 'end of each siRNAs fragment has two bases prominent. At effector steps, the siRNA duplexes trigger formation of the RNA-induced silencing complex (RISC), which is a endoribonuclease-containing complex composed of proteins and siRNA. The siRNA strands subsequently guide the RISCs to complementary mRNA molecules, where they cleave and destroy the cognate RNA. 
Design of siRNA
● 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 35% - 60% GC content is more effective. Avoid sequences that share a certain degree of homology with other related or unrelated genes. ( Some tools for designing of siRNA is OligoEngine, NEBcutter V2.0 and RNAi database. )
● In order to find the most effective siRNA sequences, the gene of interest usually needs to design several target sequences.
● 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
Chemical synthesis, digestion of long fragment dsRNA with RNase III, in vitro transcription, and expression of siRNA through the RNAi expression vector, virus vector or siRNA expression cassettes in cells has been used for preparation of siRNA.
● Chemical synthesis
21-mer DNA Oligo is used as template, and Chemical synthes is suitable for require large amounts of a defined ultrapure siRNA sequence. Drawbacks include the price and turnaround times (typically 4-12 days depending on synthesis and purification options).
● 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. It reduces the steps of detecting and screening effective siRNA sequences and saves time and money for researchers, while it may cause nonspecific gene silencing.
● In vitro transcription
29-mer DNA Oligo is used as template, and siRNAs are synthesized by in vitro transcription with 24h. The advantages is lower and 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.
● SiRNA expression vector
Most siRNA expression vectors rely on an RNA polymerase III (pol III) promoter to initiate the expression of a small hairpin siRNA 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.
● SiRNA expression cassettes
SiRNA expression cassettes (SECs) is a siRNA expression template by PCR amplification that can be introduced into cells directly -- without first being cloned into a vector. SECs is a most effective tool for screening siRNA, as it can be generated by PCR in less than a day.
SECs found to effectively elicit gene silencing can be readily cloned into a plasmid or viral vector to create an siRNA expression vecto, which then could be used for stable expression and long term studies.
SiRNA transfection
Once siRNAs or siRNA expression vectors are obtained, they must be delivered to cells. It can be introduced into eukaryotic cells by electroporation, calcium phosphate precipitation, DEAE-dextran or polybrene, mechanical method, and cationic liposome reagent. 
BrainVTA could offer the professional RNAi services to researches. If you have any needs, just contact us and our highly experienced scientists will always be ready to work you through the jungle. Meanwhile, we have prepared some coupons special for submitting clients. 


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