SMAD6 Human Pre-designed siRNA Set包括针对SMAD6 (Human)基因的不同区域设计三对siRNA，以及阴性对照、FAM 标记阴性对照和阳性对照。SMAD6全称是SMAD family member 6，Gene ID是4091。
组分与储存：
| 组分货号 | 组分名称 | 物理外观 | 1套 | 储存 |
| S1471683A | SMAD6 siRNA-1 sense and antisense (HPLC)*2 | 粉末 | 1 OD*2 (2.5 nmol*2) | -20℃. |
| S1471683B | SMAD6 siRNA-2 sense and antisense (HPLC)*2 | 粉末 | 1 OD*2 (2.5 nmol*2) | -20℃. |
| S1471683C | SMAD6 siRNA-3 sense and antisense (HPLC)*2 | 粉末 | 1 OD*2 (2.5 nmol*2) | -20℃. |
| S1471683D | siRNA Negative Control (HPLC)*2 | 粉末 | 1 OD*2 (2.5 nmol*2) | -20℃. |
| S1471683E | FAM-labeled siRNA Negative Control (HPLC)*4 | 粉末 | 0.5 OD*4 (1.25 nmol*4) | -20℃. Store in the dark. |
| S1471683F | GAPDH siRNA Positive Control (HPLC)*2 | 粉末 | 1 OD*2 (2.5 nmol*2) | -20℃. |
| S1471683G | RNase-free water | 液体 | 1 mL | -20℃. |
产品使用说明
1. 产品简介
化学合成siRNA为21~25 bp的即用型双链RNA。经过纯化、退火等处理，配置成工作浓度液体，即可直接用于转染细胞。产品为冻干粉，状态稳定。产品剂量经过严格测算标明摩尔数。本产品不保证靶基因蛋白水平的敲减。(oligo合成参数：rA分子量：329.21，rG分子量：345.21，rC分子量：305.18，rU分子量：306.17)
| siRNA(nmol) | 2.5 | 5 | 10 | 50 | 终浓度 | | 加水量 (μL) | 125 | 250 | 500 | 2500 | 20 μM | | 加水量 (μL) | 50 | 100 | 200 | 1000 | 50 μM | | 加水量 (μL) | 25 | 50 | 100 | 500 | 100 μM |
重悬 siRNA 缓冲液计算公式：终浓度（μM）= siRNA 摩尔量 (nmol) ÷ 重悬体积 (μL) × 1000
|
2. 注意事项
(1) 本产品建议在-20℃以下的环境中储存，注意避免反复冻融加速RNA降解，请分装保存。使用前瞬时离心，用RNase-free Water或灭菌ddH₂O配制成20~100μM储存液。siRNA平均分子量为13300。本产品干粉形式提供，干粉附在管壁上，打开时极易散失，所以开盖前请先离心，再小心打开管盖，溶解后盖上管盖振荡；
(2) 应当严格遵循RNA操作规则，使用RNase-free的实验耗材进行RNAi实验操作，以避免RNA降解。使用产品时最好于冰上放置；
(3) 带荧光标记的RNA，我公司会采用棕色离心管包装，因荧光标记对光敏感，必须避光保存，如 FAM、CY3、CY5 等。
3. 对照说明
| 阴性对照 negative control | 阴性对照为靶细胞无同源性的siRNA，用以排除实验中对靶基因表达量产生干扰的非siRNA干扰效应。该组干扰效率计算时该组数值计算定义为1。一个严谨的RNAi实验中，阴性对照是必不可少的。 |
| 阳性对照 GAPDH siRNA | 默认为GAPDH的干扰效率90%的siRNA，用以检测实验过程中各操作步骤的可靠性。 |
| 荧光标记阴性对照 FAM-negative control | 带荧光基团的阴性对照，检测转染效率时平行设置该组 (注意不是和其他组共转染)，避光操作，6~12h 荧光显微镜观察或流式检测siRNA的转染效率，平行设置的其他实验组的转染效率理论上与该组一致。 |
4. 实验细胞说明
转染实验中每个转染样品至少设置3个复孔；接种细胞时，每孔接种的细胞数量尽量保持一致，尽量使细胞在各孔的表面平均分布。以此降低孔间差异，保证实验的可靠性和可重复性。（以 Lipo2000转染试剂的操作参考如下，其他转染试剂参考转染试剂的说明为主）
1) 确定转染浓度
(1) 请进行梯度实验以获得细胞系转染siRNA的最佳用量。如使用几个Lipo2000的浓度，或 20~100 nM范围内改变siRNA的浓度，以确定达到最佳基因阻断水平所需要的条件。高浓度的siRNA可能具有细胞毒性；
(2) 分析的间隙更长，以使因细胞过度生长造成的细胞活性损害减少。高密度转染的细胞可能更适合优化转染条件；
(3) 荧光标记siRNA作为转染效率的指示剂，可用于优化转染条件，及时修正实验问题。
2) 转染细胞说明
贴壁细胞：转染前24h，接种0.5~2×10⁵个细胞于400μL无抗培养基中，细胞融合度为60~80%进行转染。铺板时要将细胞消化完全混匀，以避免堆积生长。悬浮细胞：转染前24 h，接种 0.5~2×10⁵个细胞于400 µL无抗培养基中，细胞数量到4~8×10⁵/孔进行转染。
3) 转染步骤
以转染试剂转染siRNA于24孔板，转染浓度为50 nM为例，其他规格容器转染请参考下表。
(1) 用50 µL Opti-MEM稀释siRNA，轻轻吹吸3~5次混匀；
(2) 颠倒混匀转染试剂，用50 µL Opti-MEM稀释1.0 µL转染试剂，轻轻吹吸3~5次混匀，室温下静置5 min；
(3) 混合siRNA和转染试剂，轻轻吹吸3~5次混匀，室温下静置20 min；
(4) 在24孔细胞板中加入转染复合物，100 µL/孔，均匀；
(5) 细胞板置于37°C、5% CO₂培养箱中培养18~48h。转染4~6h后可换新鲜培养基。
| 培养板规格 | 每孔总体积 | 培养基 | Opti-MEM (稀释 siRNA) | Opti-MEM (稀释转染试剂) | 终浓度 | siRNA 产品 * (20 μM 母液) | 转染试剂 |
| 96-well | 100 μL | 50 μL | 25 μL | 25 μL | 100 nM | 0.5 μL | 0.25 μL |
| 96-well | 100 μL | 50 μL | 25 μL | 25 μL | 50 nM | 0.25 μL | 0.25 μL |
| 96-well | 100 μL | 50 μL | 25 μL | 25 μL | 30 nM | 0.15 μL | 0.25 μL |
| 96-well | 100 μL | 50 μL | 25 μL | 25 μL | 20 nM | 0.1 μL | 0.25 μL |
| 96-well | 100 μL | 50 μL | 25 μL | 25 μL | 10 nM | 0.05 μL | 0.25 μL |
| 24-well | 500 μL | 400 μL | 50 μL | 50 μL | 100 nM | 2.5 μL | 1 μL |
| 24-well | 500 μL | 400 μL | 50 μL | 50 μL | 50 nM | 1.25 μL | 1 μL |
| 24-well | 500 μL | 400 μL | 50 μL | 50 μL | 30 nM | 0.75 μL | 1 μL |
| 24-well | 500 μL | 400 μL | 50 μL | 50 μL | 20 nM | 0.5 μL | 1 μL |
| 24-well | 500 μL | 400 μL | 50 μL | 50 μL | 10 nM | 0.25 μL | 1 μL |
| 12-well | 1 mL | 800 μL | 100 μL | 100 μL | 100 nM | 5 μL | 2 μL |
| 12-well | 1 mL | 800 μL | 100 μL | 100 μL | 50 nM | 2.5 μL | 2 μL |
| 12-well | 1 mL | 800 μL | 100 μL | 100 μL | 30 nM | 1.5 μL | 2 μL |
| 12-well | 1 mL | 800 μL | 100 μL | 100 μL | 20 nM | 1.0 μL | 2 μL |
| 12-well | 1 mL | 800 μL | 100 μL | 100 μL | 10 nM | 0.5 μL | 2 μL |
| 6-well | 2 mL | 1500 μL | 250 μL | 250 μL | 100 nM | 10 μL | 5 μL |
| 6-well | 2 mL | 1500 μL | 250 μL | 250 μL | 50 nM | 5 μL | 5 μL |
| 6-well | 2 mL | 1500 μL | 250 μL | 250 μL | 30 nM | 3 μL | 5 μL |
| 6-well | 2 mL | 1500 μL | 250 μL | 250 μL | 20 nM | 2 μL | 5 μL |
| 6-well | 2 mL | 1500 μL | 250 μL | 250 μL | 10 nM | 1 μL | 5 μL |
注：表中数据仅供参考，对于部分细胞类型的转染试剂用量可进一步优化，*表中siRNA产品取样量是20 μM储存浓度取样体积。
5. 效果检测
转染后24~72 h进行表达量检测，最佳检测时间与细胞类型、转染试剂、检测目的等相关。
(1) RNA 水平的检测：采用qPCR检测方法，siRNA转染后24~72 h即可检测到mRNA表达明显降低；
(2) 蛋白水平的检测：主要为Western Blot方法检测，检测时间受细胞内蛋白质表达量、半衰期等因素的影响，一般为48~96h；
(3) 功能筛选：应用EdU细胞增殖、EdUTP细胞凋亡等方法进行细胞功能筛选。
6. 动物实验
建议使用Chol、OMe、PS等修饰的siRNA进行实验，以延长时效。
局部给药：最直接的导入方式，siRNA 的导入效率较高，用量少，siRNA 能很快被吸收。适用于浅表器官和组织，包括眼、肌肉、皮下组织等。
系统给药：一些无法通过局部给药方式到达的靶位，如内脏、器官以及一些散列分布的靶位 (如淋巴细胞、转移性肿瘤细胞等)，可使用系统性注射方式，具有广泛的组织分布，包括心、肝、脾、肺、肾等。
7. 实验要点
(1) 保证无RNA酶环境进行实验：环境和空气中含有大量的RNA酶，痕量的RNA酶即可对RNA造成严重的降解。必须使用DEPC水或RNase-free水对RNA产品进行稀释。实验过程中，需避免 RNA产品与人体皮肤或未经去RNA酶的实验耗材接触。因呼吸产生的气体和飞沫中均含有大量的RNA酶，所以必须佩戴口罩于超净工作台内进行实验操作。
(2) 转染条件优化：优化转染条件极其重要，不同的细胞和实验中，建议都进行最佳siRNA浓度和转染试剂浓度优化。若转染试剂的转染效率不佳，可以尝试更换其他转染试剂，不同的转染试剂操作可能会有很大差异，必须严格按照说明书进行实验。有条件的话可以更换效率更高的转染方法，如电转或病毒转染。
(3) 使用多条siRNA验证表型：建议使用两条或以上的siRNA来验证表型，用于排除非靶向效应导致的假阳性结果。现在越来越多期刊也倾向于要求使用多条siRNA验证表型。
(4) 使用无抗培养基：转染6~8 h内，要使用无抗培养基。因为抗生素对细胞有毒性，会影响siRNA的递送效果。
(5) 保证健康的细胞状态：使用传代50代内的细胞进行实验，传代太多的细胞转染效率会下降。转染前必须保证细胞处于健康的状态，状态不佳的细胞其转染效率通常也很低，且对干扰效率的检测也有很大的影响。
(6) 阴性对照、阳性对照和荧光对照：使用乱序siRNA作为阴性对照，可以排除非靶向效应对实验数据的干扰。使用阳性对照 (我司赠送GAPDH siRNA)可对实验流程进行质检，帮助快速分析实验中可能存在的问题。使用荧光对照可分析siRNA的转染效率，方便进行最佳实验条件摸索。
8. 常见问题及解决方法
1) 细胞转染效率低？
(1) siRNA纯度太低，注意溶解时，避免RNA酶污染。使用经DEPC处理过的耗材和试剂。
(2) siRNA转染试剂复合物浓度低，需优化转染效率，使用合适的转染试剂剂量。
(3) 血清影响转染效率，配制混合物时，不能含有血清。培养时可以有血清，但是不要有抗生素。
(4) 脂质体转染试剂保存不当，试剂失效，应置于4℃保存。
2) 细胞转染后出现大量死亡现象？
(1) 细胞状态欠佳，需调整细胞生长状态：一般处于良好生长状态的细胞对转染试剂有更好的耐受性。
(2) siRNA或者转染试剂浓度过高。
(3) 转染试剂毒性较大，更换转染试剂。
(4) siRNA纯度低，杂质影响细胞状态。
(5) 转染时间过长，转染后未及时更换新鲜培养基。
3) 转染后，没有观察到荧光？
(1) 通常情况下，FAM转染后显示绿色荧光，CY3转染后显示红色荧光。观察时，首先确认荧光激发波长是否正确，绿光波长为495 nm，红光为550 nm。
(2) 转染后，需要立即清洗观察荧光，避免时间太久。
(3) 产品保存不当，siRNA降解，FAM淬灭。FAM容易见光分解，所以请避光保存。
(4) 转染时，要避光操作，避免FAM长时间暴露于白光下。
(5) 转染时间过长，转染后未及时更换新鲜培养基。
4) 目的基因干扰效果只有60%左右，是否判定为套餐无效？
通常套餐产品保证干扰效果能达到70%左右，如果干扰效果达到60%左右，我们也认为干扰是有意义的。如果细胞的表型及功能方面有明显变化，文献也是认可的。一般可通过提高转染效率，优化实验体系等方法，提高干扰效率。
5) 为什么要强调mRNA水平检测？可以直接检测蛋白和功能吗？
siRNA直接作用于mRNA，因此mRNA水平检测是最直接的检测指标。很多客户认为，mRNA的降解直接的结果应该是对应蛋白质含量的下降，因此蛋白水平的检测结果也可以作为有效性的检测指标。事实上，很多情况下往往出现mRNA下降水平与蛋白下降水平不对应的现象。
其可能原因有：
(1) 与选择的检测时间有关。可能因mRNA的下降尚未反映在蛋白量的变化上或者未达到足以检测的水平，所以一般建议蛋白和功能检测时间要稍微推后。
(2) 与蛋白在细胞中的表达情况有关。mRNA的翻译过程非常复杂，细胞内基因的表达总要维持一个平衡，某些蛋白表达量到一定的程度之后，足以维持其细胞功能，将可能暂时 “关闭” 表达的功能，转录出来的部分mRNA可能不参与蛋白翻译的过程，因此，mRNA水平的下调与蛋白水平下调并非完全成正相关的关系。
(3) 可能还会涉及到更加复杂的机制。
6) siRNA在细胞内可以作用多长时间？什么时候才是最好的检测时间？
siRNA介导的RNAi属于瞬时现象，不能稳定传代，一般其作用时间不可能维持很长时间，通常建议在转染后3~4 d内完成检测。最佳检测时间，因细胞、目的基因而异，多在转染后24~48 h之间检测。一般建议24~48 h检测mRNA水平，48~72 h检测蛋白水平。
7) 细胞的转染效率是否与 siRNA 序列相关？
转染效率的高低取决于细胞自身及转染方法，与siRNA的序列并没有直接关系。因此，siRNA在不同的细胞转染效率可能有差异。
8) 同样的siRNA，为什么在细胞A很有效，在细胞B则没有效果？
不同细胞的转染效率不同，基因表达水平也不一样，这些都与siRNA的作用效率有关，所以不能保证不同细胞的干扰效果一致。
9. 售后说明
如您在收到siRNA产品后一个月之内未提出异议，我们将视本制品为优良产品，不再受理投诉事宜。如确认质量问题，我们将安排免费重新合成或赔偿不超过siRNA产品本身的价值。
10. 注意事项
转染效率90%以上时保证qPCR检测mRNA水平至少一对引物有效 (首次无效后，免费设计两条)，不保证蛋白水平有效，siRNA请务必用套餐中的产品FAM-NC做转染效率评价实验，并保存白光和荧光照片。
SMAD6 Human Pre-designed siRNA Set contains three designed siRNAs for SMAD6 gene (Human), as well as a negative control, a positive control, and a FAM-labeled negative control. The full name of SMAD6 is SMAD family member 6, with a Gene ID of 4091.
Contents & Storage:
Component No.
| Component Name
| Appearance
| 1Set
| Storage
|
| S1471683A | SMAD6 siRNA-1 sense and antisense (HPLC)*2 | Powder
| 1 OD*2 (2.5 nmol*2) | -20℃. |
| S1471683B | SMAD6 siRNA-2 sense and antisense (HPLC)*2 | Powder
| 1 OD*2 (2.5 nmol*2) | -20℃. |
| S1471683C | SMAD6 siRNA-3 sense and antisense (HPLC)*2 | Powder | 1 OD*2 (2.5 nmol*2) | -20℃. |
| S1471683D | siRNA Negative Control (HPLC)*2 | Powder
| 1 OD*2 (2.5 nmol*2) | -20℃. |
| S1471683E | FAM-labeled siRNA Negative Control (HPLC)*4 | Powder
| 0.5 OD*4 (1.25 nmol*4) | -20℃. Store in the dark. |
| S1471683F | GAPDH siRNA Positive Control (HPLC)*2 | Powder
| 1 OD*2 (2.5 nmol*2) | -20℃. |
| S1471683G | RNase-free water | Liquid
| 1 mL | -20℃. |
Product Instructions
1. Product Introduction
Chemically synthesized siRNA is a ready-to-use double-stranded RNA of 21~25 bp. After purification and annealing, it is formulated into a working concentration solution for direct use in cell transfection. The product is supplied as a lyophilized powder with stable performance, and its dosage is strictly calculated and labeled in moles. This product does not guarantee knockdown of target gene protein levels. (Oligo synthesis parameters: rA molecular weight: 329.21, rG molecular weight: 345.21, rC molecular weight: 305.18, rU molecular weight: 306.17)
| siRNA (nmol) | 2.5 | 5 | 10 | 50 | Final Concentration | | Volume of Water Added (μL) | 125 | 250 | 500 | 2500 | 20 μM | | Volume of Water Added (μL) | 50 | 100 | 200 | 1000 | 50 μM | | Volume of Water Added (μL) | 25 | 50 | 100 | 500 | 100 μM |
Formula for resuspension buffer of siRNA: Final concentration (μM) = siRNA amount (nmol) ÷ Resuspension volume (μL) × 1000 |
2. Precautions
(1) This product is recommended to be stored in an environment below -20℃. Please take care to avoid repeated freeze-thaw cycles, which can accelerate RNA degradation. It is recommended to store the product in aliquots. Briefly centrifuge before use, then reconstitute with RNase-free Water or sterile ddH₂O to prepare a 20~100 μM stock solution. The average molecular weight of siRNA is 13,300. The lyophilized powder adheres to the tube wall and is easily lost when opened. Therefore, centrifuge the tube before opening the cap, then carefully remove the cap. After dissolution, cap the tube and vortex to mix.
(2) Strictly follow RNA handling protocols and use RNase-free laboratory supplies for RNAi experiments to prevent RNA degradation. It is best to keep the product on ice during use.
(3) Fluorescently labeled RNA is packaged in brown centrifuge tubes. Since fluorescent labels (e.g., FAM, CY3, CY5) are light-sensitive, they must be stored in the dark.
3. Control Instructions
| Negative control | An siRNA with no homology to the target cells, used to exclude non-siRNA interference effects on target gene expression in experiments. The value of this group is defined as 1 when calculating interference efficiency. A negative control is essential for a rigorous RNAi experiment.
|
Positive Control (GAPDH siRNA)
| An siRNA that defaults to 90% interference efficiency against GAPDH, used to verify the reliability of each step in the experimental process.
|
| FAM-Labeled Negative Control (FAM-negative control) | A negative control with a fluorescent group. This group is set in parallel to detect transfection efficiency (note: do not co-transfect with other groups). Perform operations in the dark, and observe transfection efficiency via fluorescence microscopy or flow cytometry 6~12 hours later. The transfection efficiency of other parallel experimental groups is theoretically consistent with this group.
|
4. Experimental Cell Instructions
For transfection experiments, set up at least 3 replicate wells per transfection sample. When seeding cells, ensure the number of cells per well is as consistent as possible and that cells are evenly distributed across the well surface to reduce inter-well variability and ensure experimental reliability and reproducibility. (The following is a reference for transfection with Lipo2000 reagent; for other transfection reagents, refer to their respective instructions.)
1) Dtermine Transfection Concentration
(1) Perform gradient experiments to determine the optimal siRNA dosage for your cell line. For example, test several concentrations of Lipo2000 or vary siRNA concentrations within the 20~100 nM range to identify conditions that achieve the best gene knockdown. High siRNA concentrations may be cytotoxic.
(2) Allow longer intervals between analyses to reduce cell viability damage caused by excessive cell growth. Transfecting cells at higher density may be more suitable for optimizing transfection conditions.
(3) Fluorescently labeled siRNA can be used as an indicator of transfection efficiency to optimize conditions and troubleshoot experimental issues promptly.
2) Transfection of Cells
Adherent Cells: 24 hours before transfection, seed 0.5~2×10⁵ cells in 400 μL of antibiotic-free medium. Transfect when cell confluency reaches 60~80%. Ensure cells are completely digested and mixed thoroughly during plating to avoid clumping.
Suspension Cells: 24 hours before transfection, seed 0.5~2×10⁵ cells in 400 μL of antibiotic-free medium. Transfect when the cell number reaches 4~8×10⁵ per well.
3) Transfection Procedure
The following example describes transfection of siRNA at a final concentration of 50 nM in a 24-well plate. For other plate formats, refer to the table below.
(1) Dilute siRNA in 50 μL Opti-MEM, gently pipette 3~5 times to mix.
(2) Invert the transfection reagent to mix, dilute 1.0 μL of reagent in 50 μL Opti-MEM, gently pipette 3~5 times to mix, and incubate at room temperature for 5 minutes.
(3) Combine the siRNA and transfection reagent dilutions, gently pipette 3~5 times to mix, and incubate at room temperature for 20 minutes.
(4) Add the transfection complex to the 24-well plate at 100 μL per well, ensuring even distribution.
(5) Incubate the plate at 37°C in a 5% CO₂ incubator for 18~48 hours. Fresh medium can be replaced 4~6 hours after transfection.
Culture Plate Format
| Total Volume per Well
| Culture Medium
| Opti-MEM (for siRNA dilution)
| Opti-MEM (for transfection reagent dilution)
| Final Concentration
| siRNA Product* (20 μM stock)
| Transfection Reagent
|
| 96-well | 100 μL | 50 μL | 25 μL | 25 μL | 100 nM | 0.5 μL | 0.25 μL |
| 96-well | 100 μL | 50 μL | 25 μL | 25 μL | 50 nM | 0.25 μL | 0.25 μL |
| 96-well | 100 μL | 50 μL | 25 μL | 25 μL | 30 nM | 0.15 μL | 0.25 μL |
| 96-well | 100 μL | 50 μL | 25 μL | 25 μL | 20 nM | 0.1 μL | 0.25 μL |
| 96-well | 100 μL | 50 μL | 25 μL | 25 μL | 10 nM | 0.05 μL | 0.25 μL |
| 24-well | 500 μL | 400 μL | 50 μL | 50 μL | 100 nM | 2.5 μL | 1 μL |
| 24-well | 500 μL | 400 μL | 50 μL | 50 μL | 50 nM | 1.25 μL | 1 μL |
| 24-well | 500 μL | 400 μL | 50 μL | 50 μL | 30 nM | 0.75 μL | 1 μL |
| 24-well | 500 μL | 400 μL | 50 μL | 50 μL | 20 nM | 0.5 μL | 1 μL |
| 24-well | 500 μL | 400 μL | 50 μL | 50 μL | 10 nM | 0.25 μL | 1 μL |
| 12-well | 1 mL | 800 μL | 100 μL | 100 μL | 100 nM | 5 μL | 2 μL |
| 12-well | 1 mL | 800 μL | 100 μL | 100 μL | 50 nM | 2.5 μL | 2 μL |
| 12-well | 1 mL | 800 μL | 100 μL | 100 μL | 30 nM | 1.5 μL | 2 μL |
| 12-well | 1 mL | 800 μL | 100 μL | 100 μL | 20 nM | 1.0 μL | 2 μL |
| 12-well | 1 mL | 800 μL | 100 μL | 100 μL | 10 nM | 0.5 μL | 2 μL |
| 6-well | 2 mL | 1500 μL | 250 μL | 250 μL | 100 nM | 10 μL | 5 μL |
| 6-well | 2 mL | 1500 μL | 250 μL | 250 μL | 50 nM | 5 μL | 5 μL |
| 6-well | 2 mL | 1500 μL | 250 μL | 250 μL | 30 nM | 3 μL | 5 μL |
| 6-well | 2 mL | 1500 μL | 250 μL | 250 μL | 20 nM | 2 μL | 5 μL |
| 6-well | 2 mL | 1500 μL | 250 μL | 250 μL | 10 nM | 1 μL | 5 μL |
Note: The data in the table is for reference only. The dosage of transfection reagent may need further optimization for some cell types. *The volume of siRNA product sampled is based on a 20 μM stock concentration.
5. Efficacy Detection
Perform expression level detection 24~72 hours after transfection. The optimal detection time depends on cell type, transfection reagent, and experimental objectives.
(1) RNA Level Detection: Use qPCR. A significant reduction in mRNA expression can be detected 24~72 hours after siRNA transfection.
(2) Protein Level Detection: Primarily performed via Western Blot. The detection time is influenced by intracellular protein expression levels and half-life, generally occurring 48~96 hours post-transfection.
(3) Functional Screening: Use methods such as EdU cell proliferation and EdUTP cell apoptosis assays for cell function screening.
6. In Vivo Experiments
It is recommended to use siRNA modified with Chol, OMe, PS, etc., to prolong efficacy.
Local Administration: The most direct delivery method, offering high siRNA delivery efficiency and low dosage, with rapid absorption. Suitable for superficial organs and tissues, including the eye, muscle, and subcutaneous tissue.
Systemic Administration: For target sites inaccessible via local administration, such as internal organs and diffusely distributed targets (e.g., lymphocytes, metastatic tumor cells), systemic injection can be used, providing widespread tissue distribution including the heart, liver, spleen, lungs, and kidneys.
7. Key Experimental Considerations
(1) Maintain an RNase-Free Environment: The environment and air contain abundant RNases, and even trace amounts can cause severe RNA degradation. Always dilute RNA products with DEPC-treated water or RNase-free water. Avoid contact between RNA products and human skin or non-RNase-free laboratory supplies. Since exhaled air and droplets contain high levels of RNases, experiments must be performed in a laminar flow hood while wearing a mask.
(2) Optimize Transfection Conditions: This is extremely important. For different cells and experiments, optimize the concentrations of siRNA and transfection reagent. If transfection efficiency is poor, try alternative transfection reagents, as protocols can vary significantly and must be followed strictly. If possible, use more efficient transfection methods such as electroporation or viral transfection.
(3) Validate Phenotypes with Multiple siRNAs: Use two or more siRNAs to validate phenotypes, to rule out false positives caused by off-target effects. An increasing number of journals now require this practice.
(4) Use Antibiotic-Free Medium: Use antibiotic-free medium for 6~8 hours after transfection, as antibiotics are toxic to cells and can interfere with siRNA delivery.
(5) Ensure Healthy Cell Condition: Use cells with fewer than 50 passages, as transfection efficiency declines in highly passaged cells. Cells must be in a healthy state before transfection; poor cell condition typically results in low transfection efficiency and significantly impacts interference efficiency detection.
(6) Include Negative, Positive, and Fluorescent Controls: Use scrambled siRNA as a negative control to exclude off-target effects on experimental data. Use a positive control (we provide GAPDH siRNA as a gift) to validate the experimental workflow and quickly identify potential issues. Use a fluorescent control to analyze siRNA transfection efficiency and facilitate optimization of experimental conditions.
8. Frequently Asked Questions and Troubleshooting
1) Low Cell Transfection Efficiency?
(1) Low siRNA purity: Avoid RNase contamination during resuspension. Use DEPC-treated supplies and reagents.
(2) Low concentration of siRNA-transfection reagent complex: Optimize transfection efficiency by using an appropriate dose of transfection reagent.
(3) Serum interference: Do not include serum in the mixture when preparing complexes. Serum can be present during culture, but antibiotics should be avoided.
(4) Improper storage of liposomal transfection reagent: The reagent may have expired. Store it at 4°C.
2) Massive Cell Death After Transfection?
(1) Poor cell condition: Improve cell growth status; cells in a healthy growth phase generally tolerate transfection reagents better.
(2) Excessively high concentration of siRNA or transfection reagent.
(3) High toxicity of the transfection reagent: Switch to an alternative reagent.
(4) Low siRNA purity: Impurities are affecting cell viability.
(5) Prolonged transfection time: Failure to replace medium with fresh medium in a timely manner after transfection.
3) No Fluorescence Observed After Transfection?
(1) Typically, FAM-labeled siRNA exhibits green fluorescence (excitation wavelength 495 nm), and CY3-labeled siRNA exhibits red fluorescence (excitation wavelength 550 nm). First, confirm that the correct fluorescence excitation wavelength is used.
(2) Wash cells immediately after transfection for fluorescence observation to avoid delays.
(3) Improper product storage: siRNA degradation or FAM quenching. FAM is prone to photodegradation, so store in the dark.
(4) Perform transfection in the dark to avoid prolonged exposure of FAM to white light.
(5) Prolonged transfection time: Failure to replace medium with fresh medium in a timely manner after transfection.
4) Target Gene Interference Efficiency is Only ~60%. Is the Kit Considered Ineffective?
Typically, our kit products guarantee an interference efficiency of ~70%. An efficiency of ~60% is still considered meaningful, and if significant phenotypic and functional changes are observed in cells, this is generally accepted in the literature. Interference efficiency can often be improved by increasing transfection efficiency and optimizing the experimental system.
5) Why is mRNA Level Detection Emphasized? Can Protein and Function Be Detected Directly?
siRNA acts directly on mRNA, making mRNA level detection the most direct indicator. While many assume mRNA degradation directly leads to reduced protein levels, protein detection can also be used as a validity indicator. However, mRNA and protein levels often do not correlate perfectly, due to:
(1) The timing of detection: mRNA reduction may not yet be reflected in protein levels or may not have reached a detectable threshold. Thus, protein and functional detection should generally be performed later.
(2) Protein expression dynamics: The mRNA translation process is complex. Cellular gene expression maintains homeostasis, and once certain protein levels are sufficient for function, expression may be temporarily "shut off," with some transcribed mRNA not participating in translation. Thus, mRNA downregulation does not always correlate linearly with protein downregulation.
(3) More complex underlying mechanisms may also be involved.
6) How Long Does siRNA Act in Cells? When is the Best Time for Detection?
siRNA-mediated RNAi is a transient phenomenon and cannot be stably passaged, typically lasting no longer than 3~4 days post-transfection. The optimal detection time varies by cell type and target gene, generally occurring 24~48 hours post-transfection. It is recommended to detect mRNA levels at 24~48 hours and protein levels at 48~72 hours.
7) Is Cell Transfection Efficiency Related to siRNA Sequence?
Transfection efficiency depends on the cell type and transfection method, and is not directly related to the siRNA sequence. Thus, siRNA transfection efficiency may vary across different cell types.
8) Why is the Same siRNA Effective in Cell A but Not in Cell B?
Differences in transfection efficiency and gene expression levels between cell types affect siRNA efficacy, so consistent interference efficiency across different cells cannot be guaranteed.
9. After-Sales Service
If no objections are raised within one month of receiving the siRNA product, it will be deemed to be of good quality, and no further complaints will be accepted. If a quality issue is confirmed, we will arrange for free re-synthesis or compensation not exceeding the value of the siRNA product itself.
10. Precautions
When the transfection efficiency reaches over 90%, we guarantee the validity of at least one pair of primers for mRNA level detection via qPCR (if invalid for the first time, two additional pairs will be designed free of charge). No guarantee is provided for the validity of protein level detection. For siRNA experiments, the FAM-NC included in the kit must be used to conduct transfection efficiency evaluation assays, and the bright-field and fluorescence images should be retained for record.
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