Experimental principle
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.
Experimental reagent
RNase III, DNA Oligo, Taq, dNTP, calcium chloride, phosphate buffer, reagents for cloning, siRNA synthesis kits, etc.
Experimental procedure
(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
(two) 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.
In vitro preparation
Chemical synthesis
Many foreign companies can provide high quality chemically synthesized siRNA according to user requirements. The main drawbacks include high prices and long custom cycles, especially for special needs. Since the price is higher than other methods, the cost of synthesizing 3-4 pairs of siRNAs for one gene is higher. It is more common to use other methods to screen the most efficient sequences for chemical synthesis. Best for: In the case where the most effective siRNA has been found, a large amount of siRNA is required for research. Not suitable for: long-term research such as screening siRNA, the main reason is the price factor
2. In vitro transcription
Using DNA Oligo as a template, the synthesis of siRNAs by in vitro transcription is relatively low in cost compared to chemical synthesis, and siRNAs can be obtained faster than chemical synthesis. The downside is that the scale of the experiment is limited, although an in vitro transcriptional synthesis can provide enough siRNAs to perform hundreds of transfections, but the size and amount of the reaction are always limited. And compared with chemical synthesis, it still takes a considerable amount of time for researchers. It is worth mentioning that siRNAs obtained by in vitro transcription have low toxicity, good stability and high efficiency. Only 1/10 of the amount of chemically synthesized siRNA can achieve the effect of chemical synthesis of siRNA, thus making transfection more efficient. . Most suitable for: screening siRNAs, especially when it is necessary to prepare a variety of siRNAs, when the price of chemical synthesis becomes an obstacle. Not applicable: Experiments require a large amount of a specific siRNA. Long-term research.
3. Preparation of siRNA by digesting long-segment double-stranded RNA with RNase III
A further drawback of other methods of preparing siRNA is the need to design and test multiple siRNA sequences in order to find an effective siRNA. In this way, a "mixed cocktail" of various siRNAs can be prepared to avoid this defect. A long-sequence double-stranded dsRNA was prepared by in vitro transcription using a target mRNA template, usually 200-1000 bases, and then digested with RNase III (or Dicer) to obtain a siRNAs "mixed cocktail". After removing the undigested dsRNA, the siRNA mixture can be directly transfected into cells in the same manner as a single siRNA transfection. Since there are many different siRNAs in the siRNA mixture, it is usually ensured that the gene of interest is effectively inhibited.
The main advantage of dsRNA digestion is that it can skip the steps of detecting and screening for effective siRNA sequences, saving researchers time and money (note: RNAse III is usually cheaper than Dicer). However, the shortcomings of this method are also obvious, that is, it may trigger non-specific gene silencing, especially homologous or closely related genes. Most studies now show that this usually does not affect.
Best 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, especially gene therapy in vivo. The first three methods are mainly to prepare siRNAs in vitro, and special RNA transfection reagents are needed to transfer siRNAs. in the cell. The use of siRNA expression vectors and PCR-based expression frameworks belongs to: siRNAs are transcribed in vivo from DNA templates transfected into cells. The advantage of these two methods is that there is no need to manipulate the RNA directly.
4. siRNA expression vector
Most siRNA expression vectors rely on one of three RNA polymerase III promoters (pol III) to manipulate the expression of a small hairpin RNA (shRNA) in mammalian cells. These three types of promoters include the familiar human and murine U6 promoters and the human H1 promoter. The RNA pol III promoter is used because it can express more small RNAs in mammalian cells, and it terminates transcription by adding a string (3 to 6) of U. To use such vectors, a single strand of DNA encoding a short hairpin RNA sequence is ordered, annealed, and cloned downstream of the pol III promoter of the corresponding vector. Because of the cloning involved, this process takes weeks or even months and requires sequencing to ensure that the cloned sequence is correct. The advantage of siRNA expression vectors is that longer-term studies can be performed - vectors with antibiotic markers can continue to inhibit expression of target genes in cells for weeks or longer.
Viral vectors can also be used for siRNA expression, and the advantage is that the cells can be directly and efficiently infected for gene silencing, avoiding the inconvenience caused by low efficiency of plasmid transfection, and the transfection effect is more stable. Best for: Knowing an effective siRNA sequence requires long-term gene silencing. Not applicable: Screening of siRNA sequences (in fact, mainly refers to the time-consuming and cumbersome work required for multiple clones and sequencing).
5. siRNA expression framework
siRNA expression cassettes (SECs) are siRNA expression templates obtained by PCR, including an RNA pol III promoter, a hairpin structure siRNA, and an RNA pol III termination site, which can be directly introduced into cells for expression. No need to clone into the vector beforehand. Unlike siRNA expression vectors, SECs do not require time-consuming steps such as vector cloning, sequencing, etc., and can be obtained directly from PCR, without a day. Therefore, SECs are the most effective tool for screening siRNA and can even be used to screen for optimal matching of promoters and siRNAs in a particular research system. If a restriction enzyme site is added to both ends of the PCR, the most efficient siRNA screened by SECs can be directly cloned into a vector to construct an siRNA expression vector. Constructed vectors can be used for studies that stably express siRNA and long-acting inhibition.
The main disadvantage of this method is that the 1PCR product is difficult to transfect into cells (Cytotech's Protocol can solve this problem). 2 Sequence determination cannot be performed. Misreading that may be poor in PCR and DNA synthesis cannot be found to result in unsatisfactory results. . Most suitable for: screening siRNA sequences, screening for the best promoter before cloning into a vector is not suitable for: long-term inhibition studies. (If you clone it to the carrier, you can do it)
(three) siRNA transfection
Methods for transducing prepared siRNA, siRNA expression vectors or expression frames into eukaryotic cells:
Calcium chloride, RNA (or DNA) and phosphate buffer are mixed and precipitated to form calcium phosphate particles containing minimal and insoluble calcium. The calcium phosphate-DNA complex adheres to the cell membrane and enters the cytoplasm of the target cell by pinocytosis. The size and quality of the sediment is critical to the success of calcium phosphate transfection. Each reagent used in the experiment must be carefully calibrated to ensure quality, because even one of the pHs that deviate from the optimal conditions will lead to the failure of calcium phosphate transfection.
Precautions
In order to achieve high transfection efficiency, the following points should be noted during the transfection experiment:
1. Purification of siRNA
The size and purity of the siRNA should be confirmed prior to transfection. In order to obtain high-purity siRNA, it is recommended to use glass fiber to bind, elute or remove excess nucleotides, small oligonucleotides, proteins and salt ions in the reaction by 15-20% acrylamide gel. Note: Chemically synthesized RNA usually requires gel electrophoresis (ie, PAGE gel purification).
2. Avoid RNase contamination
A small amount of RNase will cause the siRNA experiment to fail. Due to the prevalence of RNase in the experimental environment, such as skin, hair, all items that are in contact with the hand or items that are exposed to the air, it is important to ensure that each step of the experiment is not contaminated with RNase.
3. Healthy cell culture and rigorous handling to ensure repeatability of transfection
Generally, healthy cells are more efficiently transfected. In addition, a lower number of passages ensures the stability of the cells used in each experiment. In order to optimize the experiment, it is recommended to use transfected cells of less than 50 passages, otherwise the cell transfection efficiency will decrease significantly with time.
4. Avoid using antibiotics
Ambion recommends avoiding antibiotics from cell planting to 72 hours after transfection. Antibiotics accumulate toxins in penetrating cells. Some cells and transfection reagents require serum-free conditions for transfection of siRNA. In this case, a comparison experiment can be performed with both the normal medium and the serum-free medium to obtain the best transfection effect.
5. Choose the appropriate transfection reagent
The selection of good transfection reagents and optimized manipulations are critical to the success of siRNA experiments for siRNA preparation methods and target cell types.
6. Optimize transfection and detection conditions by appropriate positive controls
For most cells, housekeeping genes are a good positive control. Different concentrations of positive control siRNA were transfected into target cells (also suitable for experimental target siRNA), and the level of reduction of control protein or mRNA relative to untransfected cells was counted 48 hours after transfection. Excessive siRNA will result in cytotoxicity and even death.
7. Optimize the experiment by labeling siRNA
Fluorescently labeled siRNA can be used to analyze siRNA stability and transfection efficiency. The labeled siRNA can also be used as a siRNA intracellular localization and double labeling assay (with a labeled antibody) to track cells into which siRNA has been introduced during transfection, combining transfection with down-regulation of target protein expression.
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