如何优化GenMuteTM转染试剂，提高siRNA/DNA共转染效率？
GenMuteTM siRNA转染试剂（Cat#SL100568）是市场上最有力的siRNA传递工具之一。siRNA/DNA共转染时，siRNA的最佳浓度范围是0.5ηM到10ηM，过多的siRNA可能导致沉默效果差的“洪水效应”，切勿使用超过20ηM的siRNA。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0.5μg DNA各自加入到分别盛有50μl GenMute转染缓冲液（Cat#SL100572）的4个无菌管中进行稀释，接下来分别对4管中的siRNA进行系列稀释·····将5.0pmol siRNA加入到第一管中，2.5 pmol siRNA加入到第二管中，1.25 pmol siRNA加入到第三管中，1.25 pmol siRNA加入到第四管中。室温下静置5分钟。
3、取3.0μl GenMuteTM转染试剂，分别加入到稀释好的siRNA/DNA管中，混匀，室温下静置15分钟以形成转染复合物。请注意：室温下，转染复合物的静置时间勿超过25分钟。
4、将如上制备的转染复合物分别加入24孔板中连续的4个孔中。
5、转染24-48小时后，分别检测4孔中的沉默效果并选择出最佳的转染条件。
Optimization of GenMute™ reagent for siRNA/DNA co-transfection.
GenMute™ siRNA transfection reagent (Cat # SL100568) is one of the
most potent siRNA delivery tool in the market. The optimal siRNA
concentrations for siRNA/DNA co-transfection range from 0.5 nM to 10
nM. Excessive siRNA may lead to "flooding effect" which may comprise
silencing effect, so never use siRNA higher than 20 nM. The following
procedures will guide to optimize GenMute™ reagent for best siRNA/DNA
co-transfection 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 to each well of 24-wel plate (with serum
and antibiotics) 30 or 60 minutes before transfection.
2. Dilute 0.5 µg DNA to each sterile tube of total 4 tubes with 50 µl of GenMute transfection buffer (Cat # SL100572) followed by addition series diluted siRNA to each well of the total 4 tubes-------add 5.0 pmol siRNA to 1st tube, 2.5 pmols to 2nd tube, 1.25 pmols to 3rd tube and 1.25 pmols to 4th tube. Let's sit at RT for 5 minutes.
3. Add 3.0 µl GenMute™ reagent to diluted siRNA/DNA of each tube, 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. For 4 consecutive wells of 24-well plate, add the transfection complexes which are prepared with same concentration of DNA (0.5 µg per well) and different 4 concentrations of siRNA ranging from final 10 nM to 1.25 nM.
5. Check silencing effect in the 4 wells 24~48 hours post transfection and choose the best transfection conditions.
