RNA干扰(RNA interference，RNAi)是近年来生命科学领域最为重大的发现之一，被《Science》杂志评为2001年的十大科学进展之一，并名列2002年十大科学进展之首。它是指小分子双链 RNA可以特异性地降解同源mRNA，从而抑制或关闭特定基因表达的现象。人们只要知道了某种疾病的致病基因，就可以设计出针对该基因mRNA的小分子干扰RNA(Small interfering RNA，siRNA)，抑制或封闭该致病基因的表达，从而达到治疗疾病的目的。由于使用RNAi技术可以特异性剔除或关闭特定基因的表达，所以该技术已被广泛用于探索基因功能和传染性疾病及恶性肿瘤等基因治疗领域。RNAi是目前最为热门的生命科学研究领域，也是未来最有发展前途的新药开发领域。除此之外，RNAi技术还可以广泛应用于农业、林业、畜牧业和渔业等多种领域，进行良种培育、良种筛选和疾病治疗等。siRNA/miRNA导入细胞有以下几种方法∶化学转染技术、电穿孔法、磷酸钙共沉淀技术、显微注射和载体导入技术。由于电转的方法对细胞损伤比较大，一般不建议选择电转。化学转染技术是目前最为常用的方法，化学转染能否成功的关键在于转染试剂的选择。
siRNA/miRNA Transfection Reagent (M1505992) 是一种基于阳离子高分子聚合物的新型转染试剂，适用于siRNA和miRNA的转染。其原理为带正电的高分子聚合物与siRNA/miRNA带负电的磷酸基团形成带正电的复合物后，与细胞表面带负电的蛋白多糖相互作用，并通过内吞作用进入细胞，形成内含体。该转染试剂具有质子海绵的作用，能够吸收溶酶体中的H⁺，质子的不断流入导致复合体肿胀破裂，从而使外源siRNA或miRNA释放到细胞质中，实现siRNA/miRNA的细胞转染。
siRNA/miRNA Transfection Reagent (M1505992) 对多种常见细胞具有高水平转染效率，具有高效低毒、操作简单、重复性好等优点，适用siRNA/miRNA介导的基因抑制实验。其独特的配方使其转染后无明显细胞毒性，因此无需去除转染试剂-核酸复合物或更换新鲜培养基，也可根据具体情况优化转染体系。
产品特点：
⁕ 卓越的转染效率——针对广泛类型的细胞，均表现出优秀的转染效率，基因抑制效果好，脱靶效应低。
⁕ 细胞毒性低——作用温和，能较好地实现高转染效率与低细胞毒性之间的平衡。
⁕ 适用范围广——可用于多种细胞类型，适合siRNA/miRNA介导的基因抑制实验。
⁕ 操作简单——简单快速的实验方案，可获得稳定的结果，且转染后无需去除复合物或更换新鲜培养基。
适用范围：
siRNA/miRNA Transfection Reagent 是一种基于阳离子高分子聚合物的新型转染试剂，适用于siRNA和miRNA的转染。
实验流程概要：
图1. siRNA/miRNA Transfection Reagent (M1505992) 转染实验流程图
（以24孔板为例，其他培养板加样体积参考表一：转染量度标准）
1.准备待转染细胞
(1)贴壁细胞：转染前一天，将胰酶消化后的细胞按照每孔0.1-1x10⁵个细胞的量进行铺板，使其在转染时密度为30%-40%。
(2)悬浮细胞：转染当天，配制转染试剂-核酸复合物之前，在24孔板中进行细胞铺板，每500 µL生长培养基中加入0.5-2.5x10⁵个细胞。
⁕ 细胞状态会极大影响转染效率，待转染细胞应处于良好生长状态，建议使用生长处于指数期、存活率大于90% 的细胞进行转染。
2.准备转染试剂-核酸复合物
(1)取1 μL siRNA(20 μM，DEPC水溶解)加入到1.5 mL离心管中，加入2 μL转染试剂与siRNA混合，室温孵育3分钟。
⁕ 基因性质、细胞类型、siRNA效价、靶mRNA的半衰期和靶蛋白的周转率等因素均会影响siRNA的转染效率，建议选取siRNA作用浓度在10-100 nM之间，转染试剂的体积应根据siRNA浓度和培养皿的大小进行调整，请参见表一。
(2)往上述混合物中加入100 μL Optimal-MEM Reduced Serum Medium (with Phenol Red) (O778393)，轻轻混匀，在室温下静置30分钟，形成转染试剂-核酸复合物。
3.siRNA细胞转染
30分钟后，将上述100 µL转染试剂-核酸复合物加入每孔细胞，轻轻摇动培养板混匀，将细胞放回培养箱中继续培养。
4.分析转染细胞的基因干扰效率
转染细胞培养24-48小时后，可根据实际情况用RT-PCR、Western Blot、ELISA、荧光检测法、流式细胞术、报告基因等检测基因干扰效果，或进行后续功能实验。
⁕ 该转染试剂也适合转染miRNA，具体操作请参考siRNA的操作流程。
表一：转染量度标准
细胞培养装置规格 | 培养体系 (mL) | SiRNA(20μM) (μL) | 转染试剂 (μL) | Optimal-MEM Reduced Serum Medium (with Phenol Red) (O778393) |
96孔板
| 0.1 | 0.2 | 0.4 | 20 |
24孔板
| 0.5 | 1 | 2 | 100 |
12孔板
| 1 | 2 | 4 | 200 |
6孔板
| 2 | 4 | 8 | 400 |
60mm 皿
| 4 | 8 | 16 | 800 |
100mm 皿
| 10 | 20 | 40 | 2000 |
注意事项：
1.核酸质量：确保siRNA/miRNA是经过PAGE纯化和脱盐处理的，高纯度的siRNA/miRNA有助于获得较高的转染效率。
2. 细胞质量：细胞状态会极大影响转染效率，建议使用生长处于指数期、存活率大于90%的细胞进行转染。
3. 细胞密度：建议细胞传代后12-24 h内、细胞密度为30%-40% 时进行转染。不同的细胞转染实验，对细胞密度的要求不尽相同。在进行不同核酸或不同细胞系的转染时，需要根据说明书再次优化实验条件。此外，在实验过程中保证相同的接种条件，确保实验数据的可重复性。
4. siRNA与转染试剂使用比例：对于大多数细胞系，转染复合物中siRNA (μL of 20 μM Stock)与转染试剂(μL) 的比例在1:1至1:3之间，推荐比例为1:2。想要获得最优的基因干扰结果，需要对这一比例进行优化，根据所转染细胞及siRNA选择适合的转染比例。
5. 由于一些特殊培养基中的某些成分可能会抑制阳离子聚合物介导的转染，因此有必要检测特殊培养基与本产品的相容性。
6. 转染前，确保siRNA/miRNA基因沉默表达不会影响细胞活力。
7. 您需要设计针对目的基因的若干siRNA(小干扰RNA)，并评估其效价。
8. 整个实验过程使用无RNA酶和无热原性的材料，如离心管、枪头、缓冲液。
9. 为了您的健康安全，请规范操作，穿戴实验服与手套开展实验。
10. 本产品仅供科研使用，请勿用于临床诊断及治疗。
实验案例分析：
1.转染前一天，将稳定表达GFP的HeLa细胞接种于24孔板，使其在转染时密度为30%。
2.按照每孔计量，取1μL siRNA(GFP/NC, 20 μM)加入到1.5 mL离心管中，再加入2 μL 转染试剂 (M1505992) 与siRNA进行混合，室温孵育3分钟。
3.往上述混合物中加入100 μL Optimal-MEM Reduced Serum Medium (with Phenol Red) (O778393)，轻轻混匀，在室温条件下静置30 分钟，形成转染试剂-核酸复合物。
4.30分钟后，将上述100 µL转染试剂-核酸复合物加入每孔细胞，轻轻摇动培养板混匀，放入培养箱中继续培养。
5.siRNA转染细胞24小时后，用荧光显微镜及流式细胞仪检测GFP绿色荧光蛋白干扰效果，实验结果如图2所示。
图2. 采用 Lentivirus Packaging Transfection Reagent (S1505995) 及赛默飞RNAiMAX转染GFP siRNA 24小时后，用荧光显微镜检测(A)及流式细胞仪(B)检测GFP干扰效果。
说明：此次实验选择商业化的 siRNA转染试剂RNAiMAX作为对照组，RNAiMAX的具体实验操作按照其说明书进行。简单来说：在不含抗生素的50 μL 无血清Opti-MEM 培养基中稀释1μL siRNA(GFP, 20 μM)，轻轻混匀。然后，在不含抗生素的50 μL 无血清Opti-MEM 培养基中稀释1μL RNAiMAX，轻轻混匀。将稀释的siRNA与稀释的RNAiMAX轻轻混合，在室温下孵育20分钟。将混合物添加到细胞中，其终体积为600μL。siRNA转染细胞24小时后，用荧光显微镜及流式细胞仪检测GFP绿色荧光蛋白干扰效果。
RNA interference (RNAi) is one of the most significant discoveries in the field of life sciences in recent years. It was rated as one of the top ten scientific advances of 2001 by Science magazine and ranked first among the top ten scientific advances of 2002. It refers to the phenomenon where small double-stranded RNA can specifically degrade homologous mRNA, thereby inhibiting or silencing the expression of specific genes. As long as people know the disease-causing gene of a certain disease, they can design small interfering RNA (siRNA) targeting the mRNA of that gene to inhibit or block the expression of the disease-causing gene, thereby achieving the purpose of treating the disease. Since RNAi technology can specifically knock out or silence the expression of specific genes, it has been widely used in exploring gene functions and gene therapy fields such as infectious diseases and malignant tumors. RNAi is currently the most popular research field in life sciences and also the most promising field for new drug development in the future. In addition, RNAi technology can be widely applied in various fields such as agriculture, forestry, animal husbandry, and fishery for improved variety cultivation, variety screening, and disease treatment, etc. There are several methods for introducing siRNA/miRNA into cells: chemical transfection technology, electroporation, calcium phosphate co-precipitation technology, microinjection, and vector introduction technology. Since electroporation causes relatively great damage to cells, it is generally not recommended. Chemical transfection technology is the most commonly used method at present, and the key to the success of chemical transfection lies in the selection of transfection reagents.
siRNA/miRNA Transfection Reagent (M1505992) is a new type of transfection reagent based on cationic polymer, which is suitable for the transfection of siRNA and miRNA. Its principle is that positively charged polymers form positively charged complexes with the negatively charged phosphate groups of siRNA/miRNA. These complexes then interact with negatively charged proteoglycans on the cell surface and enter the cell through endocytosis to form endosomes. This transfection reagent has a proton sponge effect, which can absorb H⁺ in lysosomes. The continuous inflow of protons causes the complex to swell and rupture, thereby releasing exogenous siRNA or miRNA into the cytoplasm and achieving cellular transfection of siRNA/miRNA.
siRNA/miRNA Transfection Reagent (M1505992) has high transfection efficiency in various common cells, with advantages such as high efficiency, low toxicity, simple operation, and good reproducibility. It is suitable for siRNA/miRNA-mediated gene silencing experiments. Its unique formula results in no obvious cytotoxicity after transfection, so there is no need to remove the transfection reagent-nucleic acid complex or replace it with fresh medium, and the transfection system can also be optimized according to specific conditions.
Product Features:
⁕ Excellent transfection efficiency — It exhibits outstanding transfection efficiency across a wide range of cell types, with good gene silencing effects and low off-target effects.
⁕ Low cytotoxicity — It acts gently and can well balance high transfection efficiency and low cytotoxicity.
⁕ Broad application range — Suitable for multiple cell types and applicable to siRNA/miRNA-mediated gene silencing experiments.
⁕ Simple operation — The experimental protocol is simple and fast, enabling stable results. There is no need to remove the complex or replace with fresh medium after transfection.
Scope of Application:
siRNA/miRNA Transfection Reagent is a novel transfection reagent based on cationic polymer, suitable for the transfection of siRNA and miRNA.
Summary of Experimental Procedure:
Figure 1. Flowchart of transfection experiment using siRNA/miRNA Transfection Reagent (M1505992)
Experimental Procedures:
(Take a 24-well plate as an example; for the sample addition volume of other culture plates, refer to Table 1: Transfection Quantity Standards)
1. Prepare Cells for Transfection
(1) Adherent cells: One day before transfection, seed the trypsin-digested cells at a density of 0.1–1×10⁵ cells per well, ensuring the cell density reaches 30%–40% at the time of transfection.
(2) Suspension cells: On the day of transfection, before preparing the transfection reagent-nucleic acid complex, seed the cells in a 24-well plate by adding 0.5–2.5×10⁵ cells to 500 µL of growth medium per well.
⁕ Cell status significantly affects transfection efficiency. Cells to be transfected should be in good growth condition. It is recommended to use cells in the exponential growth phase with a survival rate >90% for transfection.
2. Prepare Transfection Reagent-Nucleic Acid Complex
(1) Add 1 μL of siRNA (20 μM, dissolved in DEPC water) to a 1.5 mL centrifuge tube, then add 2 μL of transfection reagent to the siRNA and mix. Incubate at room temperature for 3 minutes.
⁕ Factors such as gene properties, cell type, siRNA potency, target mRNA half-life, and target protein turnover rate can affect siRNA transfection efficiency. It is recommended to select an siRNA working concentration between 10–100 nM. The volume of transfection reagent should be adjusted according to the siRNA concentration and the size of the culture vessel; please refer to Table 1.
(2) Add 100 μL of Optimal-MEM Reduced Serum Medium (with Phenol Red) (O778393) to the above mixture, mix gently, and let stand at room temperature for 30 minutes to form the transfection reagent-nucleic acid complex.
3. siRNA Cell Transfection
After 30 minutes, add the 100 µL transfection reagent-nucleic acid complex to each well of cells. Gently swirl the plate to mix, then return the cells to the incubator for continued culture.
4. Analyze Gene Interference Efficiency in Transfected Cells
After culturing the transfected cells for 24–48 hours, the gene interference effect can be detected by methods such as RT-PCR, Western Blot, ELISA, fluorescence detection, flow cytometry, or reporter gene assays, depending on actual needs. Alternatively, proceed with subsequent functional experiments.
⁕ This transfection reagent is also suitable for miRNA transfection; please refer to the siRNA protocol for specific operations.
Table 1: Transfection Volume Standards
Specifications of Cell Culture Equipment | Culture System (mL) | SiRNA(20μM) (μL) | Transfection Reagent (μL) | Optimal-MEM Reduced Serum Medium (with Phenol Red) (O778393) |
96-well plate
| 0.1 | 0.2 | 0.4 | 20 |
24-well plate
| 0.5 | 1 | 2 | 100 |
12-well plate
| 1 | 2 | 4 | 200 |
6-well plate
| 2 | 4 | 8 | 400 |
60mm Dish
| 4 | 8 | 16 | 800 |
100mm Dish
| 10 | 20 | 40 | 2000 |
Precautions:
1. Nucleic Acid Quality: Ensure that the siRNA/miRNA has undergone PAGE purification and desalination. High-purity siRNA/miRNA helps achieve high transfection efficiency.
2. Cell Quality: Cell status significantly affects transfection efficiency. It is recommended to use cells in the exponential growth phase with a survival rate >90% for transfection.
3. Cell Density: Transfection is recommended to be performed within 12–24 hours after cell passage, when the cell density is 30%–40%. Different cell transfection experiments have different requirements for cell density. When transfecting different nucleic acids or different cell lines, it is necessary to re-optimize the experimental conditions according to the instruction manual. In addition, ensure consistent seeding conditions during the experiment to guarantee the reproducibility of experimental data.
4. Ratio of siRNA to Transfection Reagent: For most cell lines, the ratio of siRNA (μL of 20 μM Stock) to transfection reagent (μL) in the transfection complex ranges from 1:1 to 1:3, with a recommended ratio of 1:2. To obtain optimal gene silencing results, this ratio needs to be optimized, and a suitable transfection ratio should be selected based on the transfected cells and siRNA.
5. Since certain components in some special media may inhibit cationic polymer-mediated transfection, it is necessary to test the compatibility between the special medium and this product.
6. Before transfection, ensure that the silencing of siRNA/miRNA target gene expression does not affect cell viability.
7. You need to design several siRNAs (small interfering RNAs) targeting the gene of interest and evaluate their potency.
8. Use RNase-free and pyrogen-free materials (such as centrifuge tubes, pipette tips, and buffers) throughout the experiment.
9. For your health and safety, please operate in a standardized manner and wear a lab coat and gloves during the experiment.
10. This product is for research use only and shall not be used for clinical diagnosis or treatment.
Experimental Case Analysis:
1. One day before transfection, HeLa cells stably expressing GFP were seeded into a 24-well plate, ensuring the cell density reached 30% at the time of transfection.
2. For each well, add 1 μL of siRNA (GFP/NC, 20 μM) to a 1.5 mL centrifuge tube, then add 2 μL of transfection reagent (M1505992) to mix with the siRNA, and incubate at room temperature for 3 minutes.
3. Add 100 μL of Optimal-MEM Reduced Serum Medium (with Phenol Red) (Cat. No. O778393) to the above mixture, mix gently, and let it stand at room temperature for 30 minutes to form the transfection reagent-nucleic acid complex.
4. After 30 minutes, add the 100 µL transfection reagent-nucleic acid complex to each well of cells, gently swirl the plate to mix, and place it in the incubator for continued culture.
5. Twenty-four hours after siRNA transfection, the interference effect on GFP (Green Fluorescent Protein) was detected using a fluorescence microscope and flow cytometry. The experimental results are shown in Figure 2.
Figure 2. GFP interference effect detected by fluorescence microscopy (A) and flow cytometry (B) at 24 hours after transfection with GFP siRNA using Lentivirus Packaging Transfection Reagent (S1505995) and Thermo Fisher RNAiMAX.
Note: In this experiment, the commercial siRNA transfection reagent RNAiMAX was selected as the control group, and the specific experimental operation of RNAiMAX was performed according to its instruction manual. Briefly: Dilute 1 μL of siRNA (GFP, 20 μM) in 50 μL of serum-free Opti-MEM medium without antibiotics and mix gently. Then, dilute 1 μL of RNAiMAX in 50 μL of serum-free Opti-MEM medium without antibiotics and mix gently. Gently mix the diluted siRNA with the diluted RNAiMAX and incubate at room temperature for 20 minutes. Add the mixture to the cells to a final volume of 600 μL. At 24 hours after siRNA transfection, the interference effect on GFP (Green Fluorescent Protein) was detected using a fluorescence microscope and flow cytometry.
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