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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 procedure
1. Design of siRNA
1) When designing an RNAi experiment, you can first screen the target sequence at the following website:
2) Principles for selecting RNAi target sequences:
A. Starting from the AUG initiation codon of the transcript (mRNA), look for the "AA" binary sequence and note the 19 base sequence at its 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. Do not target non-translated regions (UTRs) at the 5' and 3' ends of siRNA design because these regions have abundant regulatory protein binding regions, and these UTR binding proteins or translation initiation complexes may affect the siRNP nucleic acid. The Dicer Complex binds to mRNA to affect the effect of siRNA.
B. Compare the potential sequence to the corresponding genomic database (human, mouse, rat, etc.) and exclude those sequences that are homologous to other coding sequences/ESTs.
C. Select the appropriate 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. 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:
2. Preparation of siRNA <br>The most commonly used methods have been chemical synthesis, in vitro transcription, long-segment dsRNAs by RNase III degradation in vitro, and siRNA expression vectors or viral vectors. Expression in the cells produces siRNA.
1) Preparation in vitro
A. Chemical Synthesis Many foreign companies can provide high quality chemically synthesized siRNAs 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: When the most effective siRNA has been found, a large amount of siRNA is required for research. It is not suitable for screening long-term studies such as siRNA. The main reason is the price factor.
B. 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.
C. 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 RNaseIII (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: Rapid and economical study of a gene's phenotypic phenotype does not apply to long-term research projects, or the need for a specific siRNA for research, especially gene therapy
2) In vivo expression The first three methods are mainly to prepare siRNAs in vitro, and special RNA transfection reagents are needed 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 manipulate the RNA directly.
A. siRNA expression vector Most siRNA expression vectors rely on one of three RNA polymerase III promoters (pol III) to manipulate a small hairpin RNA (shRNA) in mammalian (mammal; mammalian) cells. Expression in. These three types of promoters include the familiar human and murine U6 promoters and the human H1 promoter. The RNApolIII promoter is used because it can express more small RNAs in mammalian (mammal) cells, and it terminates transcription by adding a string (3 to 6) of U. To use such vectors, two strands of DNA encoding a short hairpin RNA sequence need to be ordered, annealed, and cloned downstream of the polIII 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).
B. siRNA expression framework
siRNA expression cassettes (SECs) are siRNA expression templates obtained by PCR, including an RNApol III promoter, a hairpin structure siRNA, and an RNA pol III termination site, which can be directly introduced into cells for expression without It was cloned 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)
3. Transfection of siRNA
There are mainly the following methods for transducing prepared siRNA, siRNA expression vector or expression framework into eukaryotic cells:
1) Calcium Phosphate Coprecipitation Calcium chloride, RNA (or DNA) and phosphate buffer are mixed and precipitated to form calcium phosphate particles containing minimal 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.
2) Electroporation electroporation transduction of molecules by exposing cells to transient high field electrical impulses. Placing the cell suspension in an electric field induces a voltage difference along the cell membrane, which is believed to cause temporary perforation of the cell membrane. Optimization of electrical pulse and field strength is important for successful transfection because excessive field strength and excessive electrical pulse time can 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
Positively charged DEAE-dextran or polybrene multimeric complexes and negatively charged DNA molecules allow DNA to bind to the cell surface. The DNA complex was introduced by osmotic shock obtained using DMSO or glycerol. Both reagents have been successfully used for transfection. DEAE-dextran is limited to transient transfection.
4) Mechanical transfection techniques also include the use of mechanical methods such as microinjection and biolistic particles. Microinjection uses a fine needle to transfer DNA, RNA or protein directly into the cytoplasm or nucleus. The gene gun uses high-pressure microprojectile to introduce macromolecules into cells.
5) Cationic liposome reagents When a cationic liposome reagent is added to water under optimized conditions, it can form minute (average size about 100-400 nm) unilamellar liposomes. These liposomes are positively charged and can be electrostatically bound to the phosphate backbone of DNA and to the surface of negatively charged cell membranes. The principle of transfection with cationic liposomes is therefore different from the previous principle of transfection with neutral liposomes. Using a cationic liposome reagent, the DNA is not pre-embedded in the liposome, but the negatively charged DNA is automatically bound to the positively charged liposome to form a DNA-cationic liposome complex. A plasmid of about 5 kb is said to bind 2-4 liposomes. The captured DNA is introduced into the cultured cells. Evidence of existing principles of DNA transduction is derived from endocytosis and lysosomes.
Guduo Bio Tips:
Purified siRNA
2. Avoid RNase contamination
3. Healthy cell culture and rigorous operation to ensure repeatability of transfection
4. Avoid using antibiotics (antibiotics)
5. Choose the appropriate transfection reagent
6. Optimize transfection and assay conditions with appropriate positive controls
7. Optimize the experiment by labeling siRNA
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 step by step in an ATP-dependent manner by cleavage by foreign sources or by various means such as transgenes, viral infections, etc. 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.