siRNA体外转染——GenMuteTM siRNA体外转染的优化
GenMuteTM siRNA转染试剂（Cat#SL100568）是市场上最有力的siRNA传递工具之一。最佳的siRNA浓度范围是1.0nM到10nM，过多的siRNA可能导致沉默效果差的“洪水效应”。我们实验室已经使用GenMuteTM转染试剂成功敲除了内源表达的生长因子。以24孔板为例，如下步骤将对如何优化GenMuteTM转染试剂做一指导：至于其他规格器皿的培养的细胞，烦请参考GenMuteTM的实验说明。
1、在转染前的30~60分钟，更换培养基，并在每孔中加入0.5ml的完全培养基（含血清和抗生素）。
2、将2.0pmol siRNA（5.0µM x 4.0µl）加入到盛有200µl GenMute转染缓冲液（Cat#SL100572）的无菌管中进行稀释，室温下静置5分钟。
3、加入2.0µl GenMute转染试剂，混匀，在室温下放置15分钟形成转染复合物。请注意：室温下，转染复合物的静置时间勿超过25分钟。
4、取50µl、25µl、12.5µl转染复合物分别加入细胞中（复孔），siRNA的终浓度各自达到10nM、5.0nM、1.25nM。
5、转染24~48小时后，检测4个不同siRNA浓度下的转染效果，选择出最佳的转染条件。
Optimization of GenMute™ reagent for siRNA silencing.
GenMute™ siRNA transfection reagent (Cat # SL100568) is one of the most potent siRNA delivery tool in the market. The optimal siRNA concentrations range from 1.0 nM to 10 nM. Excessive siRNA may lead to "flooding effect" with sub-optimal silencing. Our lab has been using GenMute™ reagent to knock down endogenously expressed growth factors with very good luck. The following procedures will guide to optimize GenMute™ reagent for best silencing in 24-well plate. For other cell culture formats, please refer to the protocol of GenMute™ reagent.
1). Change medium and add 0.5 ml of complete medium each well of 24-wel plate (with serum and antibiotics) 30 or 60 minutes before transfection.
2). Dilute 20 pmol siRNA (5.0 µM x 4.0 µl) into 200 µl of GenMute transfection buffer (Cat # SL100572) in a sterile tube and let's sit at RT for 5 minutes.
3). Add 2.0 µl GenMute reagent, briefly vortex and keep the transfection complex at RT for 15 minutes. Please note: never keep the transfection complex longer than 25 minutes at RT.
4). Add
50 µl transfection complex to your cells directly in duplicate (final 10
nM siRNA), 25 µl complex to another 2 wells (final 5.0 nM siRNA), 12.5
µl to 3rd 2 wells (final 2.5 nM siRNA) and 6.25 µl to 4th 2 wells (final
1.25 nM siRNA).
5). Check silencing effect in the 4 different siRNA concentrations 24~48 hours post transfection and choose the best transfection conditions.
