siRNA高效转染试剂是一种超高效率低毒性转染试剂，能与mRNA，siRNA等核酸形成复合物，在体外实验中，对A549、MCF-7、PC12、4T1、RAW264.7等多种难转染细胞系实现高转染效率；在体内实验中，高选择性靶向正常无肿瘤小鼠肺部并实现高效率基因表达。产品以无菌的液体形式提供。
通常情况下，使用推荐用量，每毫升本试剂约可以转染6孔板40个孔、24孔板200个孔、96孔板1000个孔，15只小鼠（体重20g）。用户指南siRNA高效转染试剂储存及运输产品4 °C密封保存；冰袋（wet ice）运输。
产品组分及储存条件：
T751606
| Components
| 100μL
| 500μL
| 1mL
| Storage
|
T751606A
| siRNA高效转染试剂
| 100μL
| 500μL
| 1mL
| 2-8℃
|
T751606B
| Transfection Buffer
| 1mL
| 5mL
| 2×5mL
| 2-8℃ |
实验材料：
• 本产品，包含siRNA高效转染试剂和Transfection Buffer两种试剂
• ⾼纯度的核酸储液（0.5∼5 μg/μL）
• ⽆⾎清培养基（如 O778393 Optimal-MEM减血清培养基 (含酚红) ）
质量控制：
• 4 °C保存条件下有效期一年
• 本产品经严格的质量检验证明，⽆⽣物污染
注意事项：
•常温下制备核酸-siRNA高效转染试剂复合物，使用灭酶灭菌管，孵育时间20～30min。
•本产品应该在4°C密闭保存，避免反复冻融。
•体外转染时，初次使用应该优化核酸浓度与siRNA高效转染试剂的比例以得到最高的转染效率。
•体外转染时，请使用适当保存和经常传代的健康细胞，并确保培养基无细菌、真菌或支原体污染。如细胞是近期复苏的液氮冻存细胞，请在转染前至少传代两次。
•体内转染时，可通过改变肿瘤体积的大小、mRNA的用量和浓度以及siRNA高效转染试剂用量对体内转染进行优化。
•转染试剂使用后请立即盖好盖子，避免长时间暴露在空气中，影响转染效率。
体外和体内转染siRNA效果示例：
图1. siRNA高效转染试剂和Lipofectamine 2000™在多种细胞系中递送siRNA的基因沉默效果。
siRNA高效转染试剂和Lipofectamine 2000™递送siRNA 48h后检测靶基因CCNB 1 mRNA(A549、Hela、MCF-7)和PTEN mRNA(4T1)相对持家基因GAPDH mRNA的表达水平（24孔板，每孔转染0.5μg siRNA）。
图2. siRNA高效转染试剂体内递送siRNA基因沉默效果。
siRNA高效转染试剂与siPTEN的组装复合物通过尾静脉注射入C57BL/6小鼠（20g）体内48h后分别检测心、脾、肾、肝和肺组织中靶基因PTEN mRNA(4T1)相对持家基因GAPDH mRNA的表达水平。肺部PTEN mRNA的相对表达水平最低，平均为0.214，沉默效果最好。
体外和体内分别转染GFP mRNA和Firefly Luciferase mRNA效果示例：
图3. siRNA高效转染试剂和Lipofectamine 2000™在多种细胞中递送mRNA的表达效果。
siRNA高效转染试剂和Lipofectamine 2000™递送GFP mRNA 48h后通过倒置荧光显微镜观察GFP表达效果（上图，比例尺100μm），通过流式细胞仪检测表达GFP的细胞的阳性率（下图）（24孔板，每孔转染1μg mRNA）。
图4. siRNA高效转染试剂体内递送mRNA基因表达效果。
siRNA高效转染试剂与Firefly Luciferase mRNA的组装复合物通过尾静脉注射入BALB/c-nu无胸腺裸鼠（20 g）体内6h后，腹腔注射200μL d-荧光素（PBS中20mg/mL），15 min后处死小鼠，分离脏器(心、脾、肾、肝、肺)，用IVIS成像系统成像。生物发光主要集中在肺部，肺部ROI值平均在10⁷左右。
体外转染时按照下表转染细胞。使用指定量的核酸试剂（siRNA、mRNA等）以及对应量的siRNA高效转染试剂。每种反应混合物体积为单个孔的体积，且考虑了移液差异。按比例计算其他孔的体积。
注：0.2μg siRNA摩尔量约为15pmol，1μg siRNA摩尔量约为75pmol，5μg siRNA摩尔量约为376pmol。
体内转染时，根据小鼠体重优化转染条件。siRNA高效转染试剂(μL)：mRNA(μg)可在3：1到6：1间调整。保证肿瘤体系在50mm³~200mm³最佳。
表1.不同体重鼠的转染用量
鼠体重
| 建议瘤体积
| siRNA高效转染试剂用量
| mRNA用量
|
15 g
| 100 mm³
| 49 μL
| 12 μg
|
20 g
| 100 mm³
| 65 μL
| 15 μg
|
25 g
| 100 mm³
| 82 μL
| 19 μg
|
30 g
| 100 mm³
| 98 μL
| 23 μg |
The High-Efficiency siRNA Transfection Reagent is an ultra-high efficiency and low toxicity transfection reagent. It can form complexes with nucleic acids such as mRNA and siRNA. In in vitro experiments, it achieves high transfection efficiency in various hard-to-transfect cell lines including A549, MCF-7, PC12, 4T1, and RAW264.7. In in vivo experiments, it selectively targets the lungs of normal tumor-free mice and enables high-efficiency gene expression. This product is supplied as a sterile liquid.
Under normal circumstances, using the recommended dosage, 1 mL of this reagent can transfect approximately 40 wells of a 6-well plate, 200 wells of a 24-well plate, 1000 wells of a 96-well plate, or be used for 15 mice (with a body weight of 20 g). User Guide: For efficient siRNA transfection reagents, store at 4°C under sealed conditions; transport using ice packs (wet ice).
Product Components and Storage Conditions:
T751606
| Components
| 100μL
| 500μL
| 1mL
| Storage
|
T751606A
| High-Efficiency siRNA Transfection Reagent
| 100μL
| 500μL
| 1mL
| 2-8℃
|
T751606B
| Transfection Buffer
| 1mL
| 5mL
| 2×5mL
| 2-8℃ |
Experimental Materials:
• This product contains two reagents: High-Efficiency siRNA Transfection Reagent and Transfection Buffer.
• High-purity nucleic acid stock solution (0.5–5 μg/μL)
• Serum-free medium (e.g., O778393 Optimal-MEM Reduced Serum Medium (with Phenol Red))
Quality Control:
• The shelf life is 1 year when stored at 4 °C.
• Strict quality inspection has confirmed that this product is free from biological contamination.
Precautions:
• Prepare the nucleic acid-High-Efficiency siRNA Transfection Reagent complex at room temperature using enzyme-free and sterile tubes, with an incubation time of 20–30 minutes.
• This product should be stored in a sealed container at 4°C and protected from repeated freeze-thaw cycles.
• For in vitro transfection, the ratio of nucleic acid concentration to High-Efficiency siRNA Transfection Reagent should be optimized during initial use to achieve the highest transfection efficiency.
• For in vitro transfection, use healthy cells that are properly preserved and regularly passaged, and ensure the medium is free from bacterial, fungal, or mycoplasma contamination. If the cells are recently revived from liquid nitrogen frozen stocks, they should be passaged at least twice before transfection.
• For in vivo transfection, in vivo transfection can be optimized by adjusting the tumor volume, the dosage and concentration of mRNA, and the dosage of High-Efficiency siRNA Transfection Reagent.
• After using the transfection reagent, tightly close the lid immediately to avoid prolonged exposure to air, which may affect transfection efficiency.
Examples of siRNA Transfection Effects in Vitro and in Vivo:
Figure 1. Gene silencing effects of siRNA delivered by the High-Efficiency siRNA Transfection Reagent and Lipofectamine 2000™ in various cell lines.
48 hours after siRNA delivery by the High-Efficiency siRNA Transfection Reagent and Lipofectamine 2000™, the relative expression levels of the target genes CCNB1 mRNA (in A549, HeLa, and MCF-7 cells) and PTEN mRNA (in 4T1 cells) compared to the housekeeping gene GAPDH mRNA were detected (24-well plate, with 0.5 μg siRNA transfected per well).
Figure 2. Gene silencing effect of siRNA delivered in vivo by the High-Efficiency siRNA Transfection Reagent.
48 hours after the assembled complex of the High-Efficiency siRNA Transfection Reagent and siPTEN was injected into C57BL/6 mice (20 g) via the tail vein, the relative expression levels of the target gene PTEN mRNA (4T1) compared to the housekeeping gene GAPDH mRNA were detected in heart, spleen, kidney, liver, and lung tissues, respectively. The relative expression level of PTEN mRNA in the lung was the lowest, with an average of 0.214, indicating the best silencing effect.
Examples of Transfection Effects of GFP mRNA and Firefly Luciferase mRNA in Vitro and in Vivo Respectively:
Figure 3. Expression effects of mRNA delivered by the High-Efficiency siRNA Transfection Reagent and Lipofectamine 2000™ in various cells.
48 hours after GFP mRNA was delivered by the High-Efficiency siRNA Transfection Reagent and Lipofectamine 2000™, the GFP expression effect was observed using an inverted fluorescence microscope (upper panel, scale bar = 100 μm), and the positive rate of cells expressing GFP was detected by flow cytometry (lower panel) (24-well plate, with 1 μg mRNA transfected per well).
Figure 4. Gene expression effect of mRNA delivered in vivo by the High-Efficiency siRNA Transfection Reagent.
Six hours after the assembled complex of the High-Efficiency siRNA Transfection Reagent and Firefly Luciferase mRNA was injected into BALB/c-nu athymic nude mice (20 g) via the tail vein, 200 μL of d-luciferin (20 mg/mL in PBS) was injected intraperitoneally. Fifteen minutes later, the mice were sacrificed, and the organs (heart, spleen, kidney, liver, and lung) were isolated and imaged using the IVIS imaging system. The bioluminescence was mainly concentrated in the lungs, with the average ROI value of the lungs being approximately 10⁷.
For in vitro transfection, transfect cells according to the table below. Use the specified amount of nucleic acid reagents (siRNA, mRNA, etc.) and the corresponding amount of High-Efficiency siRNA Transfection Reagent. The volume of each reaction mixture refers to the volume for a single well, with pipetting variations taken into account. Calculate the volumes for other wells in proportion.
Note: The molar amount of 0.2 μg siRNA is approximately 15 pmol, the molar amount of 1 μg siRNA is approximately 75 pmol, and the molar amount of 5 μg siRNA is approximately 376 pmol.
For in vivo transfection, optimize the transfection conditions according to the mouse body weight. The ratio of High-Efficiency siRNA Transfection Reagent (μL) to mRNA (μg) can be adjusted between 3:1 and 6:1. It is optimal to keep the tumor volume within 50 mm³–200 mm³.
Table 1. Transfection Dosages for Mice of Different Body Weights
Mouse Weight
| Recommended Tumor Volume
| Dosage of High-Efficiency siRNA Transfection Reagent
| Dosage of mRNA
|
15 g
| 100 mm³
| 49 μL
| 12 μg
|
20 g
| 100 mm³
| 65 μL
| 15 μg
|
25 g
| 100 mm³
| 82 μL
| 19 μg
|
30 g
| 100 mm³
| 98 μL
| 23 μg |
ZJ26F0131480
危险品化学品经营许可证（带存储）