RNA interference technology
Post-transcriptional gene silencing (PTGS) is known as RNA interference (RNAi), whereby double-stranded RNA (dsRNA) consisting of positive-sense and negative-sense RNAs corresponding to mRNAs is introduced into the cell, which can cause specific degradation of mRNAs, leading to silencing of their corresponding genes.
So far, the most commonly used methods for the preparation of siRNA include chemical synthesis, in vitro transcription, in vitro preparation of siRNA by degradation of long dsR-NA by RNAase I (e.g., Dicer, E.coli's RNAase II), and expression of siRNA in cells by siRNA expression vectors, or by viral vectors, or by expression frames of siRNAs prepared by PCR to generate siRNAs.
The first three methods are mainly for in vitro preparation of siRNA and require specialized RNA transfection reagents to transfer siRNA into cells.
In contrast, the use of siRNA expression vectors and PCR-based expression frameworks belongs to the in vivo transcription of siRNA from DNA templates transfected into cells.
The advantage of these two methods is that they do not require direct manipulation of RNA.
Instead, the following methods can be used to transfect prepared siRNA, siRNA expression vectors or expression frames into eukaryotic cells: calcium phosphate co-precipitation, electroporation, DEAE-dextran and 1,5-dimethyl-1,5-diazidecundecylmethylene polybrene, mechanical methods (e.g., microinjections and biolistic particles), and cationic cisplants (e.g., cations). particle)] and cationic liposome-mediated transfection.
Principle
The basic principle of siRNA expression cassettes is as follows: siRNA expression cassettes (SEC) are a kind of siRNA expression templates obtained by PCR, including an RNA polymerase I promoter, a hairpin siRNA and an RNA polymerase II termination site, which can be directly introduced into cells without prior cloning into vectors. can be directly introduced into cells for expression without prior cloning into a vector, as shown in the figure.
Operation method RNA interference technology Principle The basic principle of siRNA expression cassettes is that siRNA expression cassettes (SEC) are a kind of siRNA expression templates obtained by PCR, including an RNA polymerase I promoter, a hairpin siRNA, and an RNA polymerase II termination site, which can be directly introduced into cells for expression without prior cloning into vectors. introduced into cells for expression without prior cloning into vectors, as shown in the figure. The basic principle of cationic liposome-mediated transfection is the ionic interaction between the head groups of lipids, which carry a strong positive charge that neutralizes the negative charge of the DNA phosphate group, so that the cationic lipids can automatically form a monolayer shell with DNA that can be fused to the cell membrane, and thus introduce the DNA into the cell. Materials and Instruments Equipment: Move The basic process of RNAi can be divided into the following steps: (1) Upstream primer Concentration 20 μmol/L. (2) dNTP mixture Combine 100 mg each of dATP, dGTP, dCTP and dTTP sodium salts, add 2 ml of deionized water to dissolve, adjust the pH to 7.0-7.5 with 0.1 mol/L NaOH to a concentration of 5 mmol/L, and store at -20 ℃ after dispensing. Commercialized mixture (2 mmol/L each) is also available. (3) Taq DNA polymerase 5 U/μl, the final concentration of which is 1-2.5 U in a 50 μl reaction volume. (4) 10 x PCR reaction buffer 500 mmol/L KCI, 100 mmol/L Tris-HCl (pH 8.4), 15 mmol/L MgCl2. (5) 10 x D-Hanks buffer NaCl 80.0 g, Na2HPO4-2H2O 0.6 g, KCl 4.0 g, KH2PO4 0.6 g, triple-distilled water to 1000 ml, autoclaved and stored at 4 ℃. Dilute according to the proportion when using. (6) 0.25% Trypsin Trypsin 0.25 g, D-Hanks buffer added to 100 ml dissolved, filtered and sterilized, stored at 4 ℃, and rewarmed at 37 ℃ before use. (7) Serum-free medium (RPMI-1640 basic medium) 900 ml of triple-distilled water, RPMI-1640 powder 1 package (10.4 g), magnetic field stirring until completely dissolved, add NaHCO3 2 .0 g, completely dissolved and then add water to 1000 ml, filtered and sterilized. (8) Cell growth medium (RPMI-1640 growth medium) RPMI-1640 basal medium with 10% calf serum. (10) 3 mol/L CH3COONa (pH 5.2) 800 ml H2O dissolved 408.3 g CH3COONa, adjusted the pH to 5.0 with glacial acetic acid, fixed to 1 L with H2O, autoclaved. (1) Starting from the AUG start codon of the transcript (mRNA), search for the "AA" duplex sequence and write down the 19-base sequence at the 3' end of the duplex as a potential siRNA target site. Some studies have shown that siRNAs with (G+C) content around 45% to 55% are more effective than those with high (G+C) content. It is recommended not to target the untranslated regions (UTR) at the 5' and 3' ends when designing siRNAs, because there are abundant binding regions for regulatory proteins in these areas, and these UTR-binding proteins or translation initiation complexes may affect the binding of mRNAs by the siRNP endonuclease complexes, which in turn affects the effectiveness of siRNAs. (2) Compare the potential sequences with the corresponding genomic databases (human, mouse, rat, etc.) and exclude those sequences that are homologous to other coding sequences/ESTs. For example, use BLAST (www.ncbi.nlm.nih/gov/BLAST). (3) Select the appropriate target sequences to design and synthesize the downstream primers for the positive RNA and antisense RNA, respectively, at a synthesis concentration of 20 μmol/L. Usually, a gene requires the design of siRNAs for multiple target sequences and the synthesis of the corresponding primers in order to find the most effective siRNA sequence. (1) Add 5 μl of 10 x PCR buffer, 2 ul of 5 mmol/L dNTP mix, 1.5 μl of upstream primer, 1.5 μl of downstream primer (S), 1 μl of template DNA, 1 μl of Taq DNA polymerase, and 50 μl of sterilized deionized water to one Eppendorf tube; In another Eppendorf tube, add 5 μl of 10xPCR buffer, 2 μl of 5 mmol/L dNTP, 1.5 μl of upstream primer, 1.5 μl of downstream primer (AS), 1 μl of template DNA, 1 μl of Taq DNA polymerase, and make up 50 μl of sterilized deionized water. (2) A two-step amplification reaction was performed on a PCR instrument: 94 ℃, denaturation for 30 s; 72 ℃, 90 s, 35 cycles. (1) Add 1/5 volume of 3 mol/L CH3COONa and 2-fold volume of pre-cooled anhydrous ethanol to the two PCR products respectively, mix well and leave at 4 ℃ for 30 min. (2) Centrifuge the sample at 14000 g for 5 min at 4 ℃, aspirate the supernatant, then wash the precipitated DNA with 70% ethanol, centrifuge the sample again, discard the supernatant and air-dry the precipitate. (3) Dissolve the precipitated DNA in TE and measure the OD value. (1) One day before transfection, 0.25% trypsin digested the cells and counted them, and spread the cells on 35 mm cell culture dish at a density of 5x104 cells/dish, so that the density is not less than 70% on the day of transfection. Add 3 ml of serum-containing, antibiotic-free growth medium and incubate for 20-24 h at 37°C in an incubator containing 5% to 7% CO2. (2) Dilute 1.0-2.0 μg of PCR product with 100 μl of serum-free medium in a polystyrene tube. (3) Dilute 2-5 μl of Lipofectamine2000 reagent in 100 μl of serum-free medium in another polystyrene tube. after dilution, mix with the diluted PCR product within 30 min. Holding time is too long will reduce the activity. (4) Mix the diluted PCR product and diluted Lipofectamine2000, mix well and incubate at room temperature for 20 min. (5) When incubating the DNA-liposome solution, wash the cells to be transfected with serum-free medium for 3 times, then add 0.5 ml of serum-free medium to the petri dish, and incubate the tissue culture dish again in a 37°C incubator containing 5% to 7% CO2. (6) Add the complex directly into the flat dish, shake the dish and mix gently. (7) Incubate the flat dish in a 37 ℃ incubator containing 5% to 7% CO2 for 24 to 48 h without removing the complex or changing the medium, or changing the growth medium after 4 to 5 h will not reduce the transfection activity. (8) After adding the complex to the cells for 24 to 72 h, analyze cell extracts or perform in situ cell staining to detect reporter gene activity. This is dependent on cell type and promoter activity. For stable expression, cells were passaged into fresh medium 1 d after initiation of transfection and screening antibiotics were added 2 d later. Several days or weeks were required to perform stable expression. Caveat 1. Negative control should be set upA 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.It is common practice to scramble the selected siRNA sequence, again checking the results to ensure that it has no homology to other genes in the target cell. 2. Avoiding RNAase contaminationTrace amounts of RNAase will cause siRNA experiments to fail. Since RNAase is prevalent in the experimental environment, such as skin, hair, all objects touched with bare hands or exposed to the air, it is important to ensure that each step of the experiment is free from RNAase contamination. 3. Healthy cell cultures and strict handling ensure reproducibility of transfectionIn general, 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 less than 50 generations of transfected cells, otherwise the efficiency of cell transfection will decrease significantly over time. 4. Avoid the use of antibioticsAvoid the use of antibiotics from the time the cells are planted until 72h after transfection. Antibiotics accumulate toxins in the penetrating cells. Some cells and transfection reagents require serum-free conditions during siRNA transfection.In this case, comparison experiments can be done with both normal medium and serum-free medium to get the best transfection results. 5. Optimize transfection and detection conditions by suitable positive controlsFor most cells, the housekeeping gene is a better positive control. Transfect different concentrations of positive control siRNA into target cells (also suitable for experimental target siRNA), and count the reduced level of control protein or mRNA relative to untransfected cells after 48h of transfection. Excessive siRNA will lead to cytotoxicity to the point of death. 6. Optimize experiments by labeling siRNAsFluorescently labeled siRNA can be used to analyze siRNA stability and transfection efficiency. Labeled siRNAs can also be used for intracellular localization of siRNAs and double-labeling experiments (with labeled antibodies) to track cells introduced with siRNAs during transfection, combining transfection with down-regulation of target protein expression. For more product details, please visit Aladdin Scientific website.
①PCR instrument, (sterilized)
② cell culture incubator, constant temperature water bath box
③ ultraviolet spectrophotometer
④Microscope
⑤35 mm cell culture dish
⑤35 mm cell culture dish ⑥Freezing centrifuge
⑦0.2 ml and 1.5 ml polystyrene Eppendorf tubes
Reagents:
①Materials: exponentially growing mammalian cell cultures
② PCR template for siRNA expression framework, upstream primers, dNTP mix, Taq DNA polymerase, 10 x PCR reaction buffer
③Lipofectamine 2000
④Cell growth medium, serum-free medium, 0.25% trypsin
⑤3 mol／L CH
⑤ 3 mol／L CH
COONa (pH 5.2) ⑥TE (pH 8.2)
⑥ TE (pH 8.0)
⑦ Anhydrous ethanol, 70% ethanol