GenMuteTM siRNA轉染試劑（Cat#SL100568）是市場上最有力的siRNA傳遞工具之一。最佳的siRNA濃度范圍是1.0nM到10nM，過多的siRNA可能導致沉默效果差的“洪水效應”。我們實驗室已經使用GenMuteTM轉染試劑成功敲除了內源表達的生長因子。以24孔板為例，如下步驟將對如何優化GenMuteTM轉染試劑做一指導：至于其他規格器皿的培養的細胞，煩請參考GenMuteTM的實驗說明。
1、在轉染前的30~60分鐘，更換培養基，并在每孔中加入0.5ml的完全培養基（含血清和抗生素）。
2、將2.0pmol siRNA（5.0μM x 4.0μl）加入到盛有200μl GenMute轉染緩沖液（Cat#SL100572）的無菌管中進行稀釋，室溫下靜置5分鐘。
3、加入2.0μl GenMute轉染試劑，混勻，在室溫下放置15分鐘形成轉染復合物。請注意：室溫下，轉染復合物的靜置時間勿超過25分鐘。
4、取50μl、25μl、12.5μl轉染復合物分別加入細胞中（復孔），siRNA的終濃度各自達到10nM、5.0nM、1.25nM。
5、轉染24~48小時后，檢測4個不同siRNA濃度下的轉染效果，選擇出最佳的轉染條件。
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.