The steps include:
(1) Design of siRNA
1. When designing RNAi experiments, you can first select the target sequence on the following website:
Genesil
Ambion
2. Selection principle of RNAi target sequence:
(1) Starting from the AUG start code of the transcript (mRNA), look for the "AA" tandem sequence and write down the 19-base sequence at its 3 end as a potential siRNA target site. Some research results show that siRNA with GC content of 45% -55% is more effective than those with high GC content.
Tuschl and others suggest that when designing siRNAs, do not target untranslated regions (UTRs) at the 5 and 3 ends, because these places are rich in regulatory protein binding regions, and these UTR binding proteins or translation initiation complexes may affect siRNP The endonuclease complex binds to mRNA and thus affects the effect of siRNA.
(2) Compare the potential sequences with the corresponding genomic database (human, or mouse, rat, etc.) and exclude those homologous to other coding sequences / EST.
For example using BLAST
(3) Select the appropriate target sequence for synthesis. Usually a gene needs to design multiple target sequence siRNA to find the most effective siRNA sequence.
3. Negative control
A complete siRNA experiment should have a negative control. The siRNA used as a negative control should have the same composition as the selected siRNA sequence, but no obvious homology to the mRNA. The usual practice is to scramble the selected siRNA sequence, and also check the results to ensure that it has no homology with other genes in the target cell.
4. Currently confirmed siRNA can be found on the following web page:
Ambion
(2) Preparation of siRNA
The most commonly used methods so far include chemical synthesis, in vitro transcription, long-length dsRNAs prepared by RNase III degradation (eg Dicer, E. coli, RNase III) in vitro preparation of siRNA, and siRNA expression vector or viral vector, PCR prepared siRNA The expression cassette is expressed in the cell to produce siRNA.
In vitro preparation
Chemical synthesis
Many foreign companies can provide high-quality chemically synthesized siRNA according to user requirements. The main disadvantages include high prices and long customization cycles, especially those with special needs. Because the price is higher than other methods, the cost of synthesizing 3-4 pairs of siRNAs for a gene is even higher. The more common method is to use other methods to screen out the most effective sequence and then perform chemical synthesis.
Best for: When 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, mainly due to price factors
2. In vitro transcription
Using DNA Oligo as a template, siRNAs are synthesized by in vitro transcription, the cost is relatively low compared to chemical synthesis, and siRNAs can be obtained faster than chemical synthesis. The disadvantage is that the scale of the experiment is limited. Although one in vitro transcription synthesis can provide enough siRNAs for hundreds of transfections, the scale and amount of the reaction are always limited. And compared to chemical synthesis, it still takes a considerable amount of time for researchers. It is worth mentioning that the 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 that chemically synthesized siRNA can achieve, thereby making transfection more efficient .
Best for: Screening siRNAs, especially when multiple siRNAs need to be prepared, and the price of chemical synthesis becomes an obstacle.
Not applicable: The experiment requires a large amount of a specific siRNA. Long-term research.
3. Digest long double-stranded RNA with RNase III to prepare siRNA
The disadvantage 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, this defect can be avoided by preparing a "mixed cocktail" mixed with various siRNAs. Select a target mRNA template that is usually 200-1000 bases, prepare long double-stranded dsRNA by in vitro transcription, and then digest in vitro with RNase III (or Dicer) to obtain a "mixed cocktail" of siRNAs. After removing the undigested dsRNA, this siRNA mixture can be directly transfected into cells, just like single siRNA transfection. Because there are many different siRNAs in the siRNA mixture, it can usually ensure that the target gene is effectively suppressed.
The main advantage of dsRNA digestion is that it can skip the steps of detecting and screening 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 cause non-specific gene silencing, especially homologous or closely related genes. Most studies now show that this situation usually has no impact.
Best for: Quickly and economically studying the phenotype of a gene's loss of function.
Not suitable for: long-term research projects, or the need for a specific siRNA for research, especially gene therapy.
In vivo expression
The first three methods are mainly for preparing siRNAs in vitro, and require special RNA transfection reagents to transfer siRNAs into cells. 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 directly manipulate RNA.
4. siRNA expression vector
Most siRNA expression vectors rely on one of the three RNA polymerase III promoters (pol III) to manipulate the expression of a short hairpin RNA (shRNA) in mammalian cells. These three types of promoters include the familiar human and mouse U6 promoters and human H1 promoters. The reason why the RNA pol III promoter is used is because it can express more small-molecule RNA in mammalian cells, and it terminates transcription by adding a string (3 to 6) of U. To use this type of vector, you need to order 2 single strands of DNA encoding a short hairpin RNA sequence, annealed, and cloned into the corresponding vector downstream of the pol III promoter. Since cloning is involved, this process takes weeks or even months, and it also needs to be sequenced to ensure that the cloned sequence is correct.
The advantage of siRNA expression vectors is that they can be studied for a longer period of time-vectors marked with antibiotics can continue to suppress the expression of target genes in cells for weeks or even longer.
Viral vectors can also be used for siRNA expression. The advantage is that they can directly and efficiently infect cells to conduct gene silencing research, avoid all the inconveniences caused by low plasmid transfection efficiency, and the transfection effect is more stable.
Best for: Knowing a valid siRNA sequence requires maintaining gene silencing for a longer period of time.
Not applicable: screening siRNA sequences (in fact, it mainly refers to the time-consuming and tedious work that requires 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. There is no need to clone into the vector beforehand. Unlike siRNA expression vectors, SECs do not require time-consuming steps such as vector cloning and sequencing, and can be obtained directly by PCR, instead of one day. Therefore, SECs have become the most effective tool for screening siRNA, and can even be used to screen the most suitable combination of promoter and siRNA in a specific research system. If the restriction sites are added at both ends of PCR, the most effective siRNA selected by SECs can be directly cloned into the vector to construct the siRNA expression vector. The constructed vector can be used for the study of stable expression of siRNA and long-term inhibition.
The main disadvantages of this method are:
â‘ The PCR product is difficult to transfect into the cell (The protocol of Crystal Corporation can solve this problem)
â‘¡Sequencing cannot be performed, and misunderstandings that may be poor during PCR and DNA synthesis cannot be found, resulting in unsatisfactory results.
Best for: Screening siRNA sequences, screening for the best promoter before cloning into a vector
Not applicable: Long-term inhibition studies. (If it is cloned into the vector, it will be fine)
(3) siRNA transfection
There are mainly the following methods for transducing prepared siRNA, siRNA expression vector or expression framework into eukaryotic cells:
1. Co-precipitation of calcium phosphate
Calcium chloride, RNA (or DNA) and phosphate buffer are mixed and precipitated to form very small insoluble calcium phosphate particles containing DNA. The calcium phosphate-DNA complex adheres to the cell membrane and enters the cytoplasm of the target cell through endocytosis. The size and quality of the precipitate is critical to the success of calcium phosphate transfection. Each reagent used in the experiment must be carefully calibrated to ensure quality, because even a pH that deviates from the optimal condition by one tenth will cause the failure of calcium phosphate transfection.
2. Electroporation
Electroporation transduces molecules by exposing cells to brief high-field electric pulses. Placing the cell suspension in an electric field induces a voltage difference along the cell membrane, which is thought to cause temporary perforation of the cell membrane. The optimization of electric pulse and field strength is very important for successful transfection, because too high field strength and long electric pulse time will irreversibly damage the cell membrane and lyse cells. In general, successful electroporation processes are accompanied by high levels (50% or higher) of toxicity.
3. DEAE-dextran and polybrene
The positively charged DEAE-dextran or polybrene polymer complex and negatively charged DNA molecules allow DNA to bind to the cell surface. The DNA complex is introduced by osmotic shock obtained using DMSO or glycerin. Both reagents have been successfully used for transfection. DEAE-dextran is limited to transient transfection.
4. Mechanical method
Transfection techniques also include the use of mechanical methods, such as microinjection and biolistic particles. Microinjection uses a thin needle to transfer DNA, RNA or protein directly into the cytoplasm or nucleus. The gene gun uses high-pressure microprojectiles to introduce large molecules into cells.
5. Cationic liposome reagent
When the cationic liposome reagent is added to water under optimized conditions, it can form tiny (average size about 100-400 nm) monolayer liposomes. These liposomes are positively charged and can bind to the phosphate backbone of DNA and the surface of negatively charged cell membranes by electrostatic action. Therefore, the principle of transfection using cationic liposomes is different from that of neutral liposomes. With cationic liposome reagents, DNA is not embedded in liposomes in advance, but negatively charged DNA automatically binds to positively charged liposomes to form a DNA-cationic liposome complex. It is said that a plasmid of about 5 kb will bind 2-4 liposomes. The captured DNA will be introduced into the cultured cells. Existing evidence for the principle of DNA transduction comes from endosomes and lysosomes.
In order to achieve high transfection efficiency, during the transfection experiment, it is necessary to pay attention to the following points:
1. Purified siRNA
Confirm the size and purity of siRNA before transfection. In order to obtain high-purity siRNA, it is recommended to use glass fiber binding, elution or 15-20% acrylamide glue to remove excess nucleotides, small oligonucleotides, proteins and salt ions in the reaction. Note: Chemically synthesized RNA usually requires gel electrophoresis purification (ie PAGE gel purification).
2. Avoid RNase contamination
A small amount of RNase will cause the siRNA experiment to fail. Since RNases are common in the experimental environment, such as skin, hair, all items that have been touched with bare hands or exposed to the air, it is very important to ensure that each step of the experiment is not contaminated with RNases.
3. Healthy cell culture and strict operation ensure the repeatability of transfection
In general, the transfection efficiency of healthy cells is higher. In addition, the lower number of passages can ensure the stability of the cells used in each experiment. In order to optimize the experiment, it is recommended to use transfected cells below 50 generations, otherwise the cell transfection efficiency will decrease significantly with time.
4. Avoid antibiotics
Ambion recommends avoiding antibiotics from the time of cell planting to 72 hours after transfection. Antibiotics can accumulate toxins in the penetrated cells. Some cells and transfection reagents require serum-free conditions when transfecting siRNA. In this case, the normal medium and serum-free medium can be used for comparison experiments to obtain the best transfection effect.
5. Choose the appropriate transfection reagent
According to the different methods of siRNA preparation and target cell types, the selection of good transfection reagents and optimized operations are critical to the success of siRNA experiments.
6. Optimize transfection and detection conditions with appropriate positive controls
For most cells, housekeeping genes are a good positive control. Different concentrations of positive control siRNA were transferred into target cells (also suitable for experimental target siRNA), and the reduction level of control protein or mRNA relative to untransfected cells was counted 48 hours after transfection. Too much siRNA will cause 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 for intracellular localization of siRNA and double-labeling experiments (with labeled antibodies) to track cells introduced with siRNA during transfection, combining transfection with down-regulation of target protein expression.
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