First, the molecular mechanism of RNAi
Biochemical and genetic studies have shown that RNA interference includes inititation and effector steps. At the initial stage, the added small RNA was cleaved into small interfering RNAs (siRNAs) of 21-23 nucleotides in length. Evidence suggests that an enzyme called Dicer is a member of the RNase III family that specifically recognizes double-stranded RNA. It can be introduced in an ATP-dependent manner by exogenous introduction or by transgenic, viral infection, etc. Double-stranded RNA, cleavage degrades RNA into 19-21 bp double-stranded RNAs (siRNAs) with 2 bases at the 3' end of each fragment.
In the RNAi effect phase, the siRNA duplex binds to a ribozyme complex to form a so-called RNA-induced silencing complex (RISC). Activating RISC requires an ATP-dependent process of de-sequencing small RNAs. The activated RISC is mapped to homologous mRNA transcripts by base pairing and cleaves mRNA at a position 12 bases from the 3' end of the siRNA. Although the exact mechanism of cleavage is not known, each RISC contains an siRNA and an RNase different from Dicer.
In addition, studies have shown that dsRNA containing a promoter region is also cleaved into a 21-23 nt fragment in a plant. This dsRNA can methylate the endogenous corresponding DNA sequence, thereby rendering the promoter incapable of functioning. Downstream gene silencing.
Second, how to carry out RNAi test
(1) Design of siRNA
1. When designing an RNAi experiment, you can first filter the target sequence at the following website:
Http://
Http://
http://
http://design.dharmacon.com/rnadesign/default.aspx?SID=45358710
2. Principles for selecting RNAi target sequences:
(1) Starting from the AUG initiation code of the transcript (mRNA), look for the "AA" sequence and note the 19 base sequence at the 3' end as a potential siRNA target site. Studies have shown that siRNA with GC content between 45% and 55% is more effective than those with higher GC content.
Tuschl et al. suggested not to target non-translated regions (UTRs) at the 5' and 3' ends when designing siRNAs because these regions have abundant regulatory protein binding regions, and these UTR-binding proteins or translation-initiating complexes may The effect of the siRNP endonuclease complex binding mRNA on the siRNA is affected.
(2) Compare the potential sequence to the corresponding genomic database (human, mouse, rat, etc.) and exclude those sequences homologous to other coding sequences/ESTs.
For example using BLAST ( )
(3) Select a suitable target sequence for synthesis. Usually a gene needs to design multiple target sequence siRNAs to find the most efficient siRNA sequence.
3. Negative control
A complete siRNA assay should have a negative control, and the siRNA as a negative control should have the same composition as the selected siRNA sequence, but no significant homology to the mRNA. It is common practice to scramble the selected siRNA sequence and also check the results to ensure that it has no homology to other genes in the target cell of interest.
4. Currently confirmed siRNAs can be found on the following webpage:
Http://design.dharmacon.com/catalog/category.aspx?key=49
Http://
http://web.mit.edu/mmcmanus/
http://python.penguindreams.net/Order_Entry/jsp/BrowseCatalog.jsp?Category=Published
b) Preparation of siRNA
The most commonly used methods have been the preparation of siRNA by chemical synthesis, in vitro transcription, long-range dsRNAs by RNase III degradation (eg Dicer, E. coli, RNase III), and siRNA prepared by siRNA expression vector or viral vector. Expression cassettes are expressed in cells to produce siRNA.
1. Chemical synthesis of siRNA
Advantages: Convenience, the researcher does not need to do any work.
Disadvantages: the price is more expensive, the efficiency is only 1/10-1/40 of the transcript-synthesized shRNA, the gene inhibition duration is short, the cytotoxicity is large, the transfection efficiency is low, and the method has irreparable defects in the synthesis process. It is impossible to synthesize shRNA, and it is impossible to correct about 20% of base errors generated in the synthesis.
Applicable to: It is recommended that this synthesis method no longer be used.
Not applicable: Basically not applicable to all current laboratories.
Evaluation: ☆☆☆☆☆
2. Biosynthetic transcriptional production coding siRNA
Advantages: low price, high inhibition efficiency, low concentration of siRNA can achieve inhibition. In vitro transcription synthesis, relatively close to physiological state.
Disadvantages: The effectiveness cannot be guaranteed, the scale of the experiment is limited, a large amount of production cannot be performed, the long-term suppression effect cannot be maintained, and the user is required to participate in the operation, and it is difficult to ensure the success of the experiment.
Applicable to: Screening for siRNAs in particular requires the preparation of multiple siRNAs, taking into account the synthetic price.
Not applicable: Experiments require a large amount of a specific siRNA. Long-term research.
Evaluation: ★★★☆☆
Although some companies have introduced shRNA and siRNA kits, most of these kits are easy to operate. After all, RNA operations are inevitable, and users will inevitably have many unexpected difficulties. Crystal Games can solve your worries completely. You can directly Get a valid siRNA or shRNA sequence.
3. Dicer enzymatic production of siRNA
Advantages: The steps of screening and detecting effective siRNA sequences can be skipped, saving time and expense.
Disadvantages: It is possible to trigger non-specific gene silencing, especially homologous or closely related genes.
Suitable for: quickly and economically studying the phenotype of a gene's loss of function
Not suitable for: long-term research projects, or need a specific siRNA for research
Evaluation: ★★★☆☆
4. Vector encoding shRNA for siRNA
Advantages: The preparation quality is controllable, and the antibiotic-labeled vector can continuously inhibit the expression of the target gene in the cell for several weeks or more, and can be expanded in a large amount.
Disadvantages: The correct folding rate of shRNA after transcription is not guaranteed, which may result in non-specific inhibition. The transfection efficiency of plasmid vector is unstable, which is related to cell type.
Applicable to: Know a valid siRNA sequence, need to maintain gene silencing for a long time, or need to use antibiotics to screen cells that can express siRNA. Long-term research.
Not suitable for: screening siRNA sequences (in fact, mainly refers to the time-consuming and cumbersome work required for multiple cloning and sequencing)
Evaluation: ★★★★☆
CrystalScience has a patented technology for the production of siRNAs by shRNA-encoding vectors, which can encode up to six target gene siRNA sequences at a time, saving you screening time and ensuring the effectiveness of siRNA. Specific details: http://#6
5. Viral particle production shRNA
Advantages: easy to prepare, purify, concentrate, wide range of host, high infection rate, stable physical and chemical properties, unconformed host genome, low genotoxicity and immunotoxicity, is one of the best RNA interference technologies.
Disadvantages: It takes a long time, even if it is the fastest, it takes about 60 days. It requires higher experimental conditions and smaller capacity, which may be accompanied by partial inflammatory reaction.
Applicable to: low transfection efficiency can not be solved, need to maintain a short time gene silencing siRNA
Not suitable for: large-scale screening of siRNA or long-term research, mainly due to price factors
Evaluation: ★★★★★
Crystal Games has an unpublished virus preparation technology that can complete shRNA virus preparation within 15 days. The price is close to other methods of shRNA preparation, and it is very likely to replace the conventional virus expression shRNA method as the best RNA interference technology.
6. PCR preparation of shRNA expression cassette for production of siRNA
Advantages: low cost, convenient and fast, can be used for screening of effective sequences, and can be used for plasmid vector construction.
Disadvantages: low transfection efficiency, not suitable for large-scale preparation.
Suitable for: screening siRNA sequences, screening the best promoter before cloning into the vector
Not applicable: Long-term inhibition studies. (If you clone it to the carrier, you can do it)
Evaluation: ★★★☆☆
The reference mode recommended by Crystal Games for your RNAi experimental method:
1. Establish target genes that require interference
2. Check the transfection of cells used
You can choose a plasmid of about 4kb size and an executable transfection method to detect transfection efficiency.
Transfection efficiency is over 40%: any RNA interference method can be chosen.
Transfection efficiency is between 10% and 40%: in vitro synthesized si RNA or shRNA plasmid expression vector with selection marker can be used.
Transfection efficiency is less than 10%: shRNA plasmid expression vector or viral vector with a selection marker.
3. Si RNA sequence selection design
Prepare 20-30 si RNA sequences, select valid sequences or commission a specialized company (eg, crystal game organisms) design (3 sequences guarantee 1 valid)
4. Start experiment
Third, the application prospects of RNAi
1. New tools for studying gene function
Studies have shown that RNAi can inactivate or reduce the expression of specific genes in mammals, produce a variety of phenotypes, and the time to inhibit gene expression can be controlled at any stage of development, producing a similar knock-out effect. The entire genome sequence of C. elegans and Drosophila has been tested and a large number of new genes with unknown functions have been discovered. RNAi will greatly facilitate the study of the function of these new genes. Compared with traditional gene knockout technology, this technology has the advantages of less investment, short cycle, and simple operation. Recently, the reports of RNAi successfully used to construct transgenic animal models are increasing, which indicates that RNAi will become an indispensable function of research genes. Tool of.
2. New ways to study signaling pathways
The combination of traditional deletion mutation technology and RNAi technology can easily determine the upstream and downstream relationship of different genes in complex signaling pathways. Clemensy et al. applied RNAi to study insulin signaling pathways in Drosophila cell lines, and obtained insulin with known insulin. The relationship between DSH3PX1 and DACK was analyzed based on the complete agreement of the information transmission pathway. It was confirmed that DACK is an upstream kinase located in DSH3PX1 phosphorylation. RNAi technology is simpler, faster and more reproducible than traditional transfection experiments. Overcome the turn
In the dyeing experiment, the specific aggregation and transfection efficiency of recombinant protein are not high, so it is considered that RNAi technology may become a new way to study cell signaling pathway.
3. New strategies for gene therapy
RNAi has the functions of resisting viral invasion, inhibiting transposon activity, and preventing excessive proliferation of selfish gene sequences. Therefore, RNAi can be used to produce antiviral plants and animals, and dsRNAs corresponding to highly homologous segments in different viral transcription sequences can be utilized. Resist against multiple viruses.
Tumors are the result of gene network regulation of multiple gene interactions. The blocking of a single oncogene induced by traditional techniques cannot completely inhibit or reverse the growth of tumors, and RNAi can utilize multiple genes of the same gene family to have a homology. The highly conserved sequence is designed to target dsRNA molecules of this segment sequence. Injecting only one dsRNA can produce simultaneous deletion of multiple genes, or multiple dsRNAs can be injected simultaneously to unequate multiple sequences. The genes are simultaneously eliminated.
Although the current application of RNAi technology in mammals is still in the exploratory stage, its successful application in vertebrates such as zebrafish and mice indicates that RNAi will become an important component of gene therapy, artificially synthesized dsRNA oligomeric drugs. Development will likely become an emerging industry with great development prospects.
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