High throughput RNAi assay optimization using adherent cell cytometry.
High throughput RNAi assay optimization using adherent cell cytometry.
复制标题
DOI：
10.1186/1479-5876-9-48
复制
发表时间：
2011-04-25
影响因子：
7.4
通讯作者：
Logerfo FW
中科院分区：
文献类型：
--
作者：
Nabzdyk CS;Chun M;Pradhan L;Logerfo FW
siRNA技术是一种很有前途的血管疾病基因治疗工具。由于试剂和细胞类型的多样性，RNAi实验优化可能非常耗时。在这项研究中，贴壁细胞流式细胞术用于快速优化siRNA转染人主动脉血管平滑肌细胞（AoSMC）。将AoSMC以3000 - 8000个细胞/孔的密度接种在96孔板中。24小时后，使用无转染试剂HiPerfect或Lipofectamine RNAiMax，用非靶向未标记的siRNA（50 nM）或非靶向标记的siRNA siGLO Red（5或50 nM）转染AoSMC。对于细胞计数，使用Hoechst核染色或Cell Tracker绿色。对于数据分析，使用贴壁细胞计数器Celigo®。将数据标准化为单独转染试剂组，并表示为红色像素计数/细胞。24小时后，没有转染条件导致细胞损失。将红色荧光计数标准化为AoSMC计数。与HiPerfect或无转染试剂相比，RNAiMax在5 nM siGLO Red（4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13红色像素/细胞）和50 nM siGLO Red（6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19）下更有效。荧光表达结果支持在AoSMC中通过使用MARCKS靶向siRNA实现的基因敲除。这项研究强调，RNAi递送在很大程度上取决于递送方法的选择。贴壁细胞计数法可用作优化RNAi测定的高通量筛选工具。该技术可以加速体外细胞测定，从而节省成本。
siRNA technology is a promising tool for gene therapy of vascular disease. Due to the multitude of reagents and cell types, RNAi experiment optimization can be time-consuming. In this study adherent cell cytometry was used to rapidly optimize siRNA transfection in human aortic vascular smooth muscle cells (AoSMC). AoSMC were seeded at a density of 3000-8000 cells/well of a 96well plate. 24 hours later AoSMC were transfected with either non-targeting unlabeled siRNA (50 nM), or non-targeting labeled siRNA, siGLO Red (5 or 50 nM) using no transfection reagent, HiPerfect or Lipofectamine RNAiMax. For counting cells, Hoechst nuclei stain or Cell Tracker green were used. For data analysis an adherent cell cytometer, Celigo® was used. Data was normalized to the transfection reagent alone group and expressed as red pixel count/cell. After 24 hours, none of the transfection conditions led to cell loss. Red fluorescence counts were normalized to the AoSMC count. RNAiMax was more potent compared to HiPerfect or no transfection reagent at 5 nM siGLO Red (4.12 +/-1.04 vs. 0.70 +/-0.26 vs. 0.15 +/-0.13 red pixel/cell) and 50 nM siGLO Red (6.49 +/-1.81 vs. 2.52 +/-0.67 vs. 0.34 +/-0.19). Fluorescence expression results supported gene knockdown achieved by using MARCKS targeting siRNA in AoSMCs. This study underscores that RNAi delivery depends heavily on the choice of delivery method. Adherent cell cytometry can be used as a high throughput-screening tool for the optimization of RNAi assays. This technology can accelerate in vitro cell assays and thus save costs.
登录
查看更多内容
影响因子：
4.8
作者：
Monahan, Thomas S.;Andersen, Nicholas D.;LoGerfo, Frank W.
通讯作者：
LoGerfo, Frank W.
ADAMTS-7 介导球囊损伤大鼠动脉中的血管平滑肌细胞迁移和新内膜形成
影响因子：
20.1
作者：
Wang, Li;Zheng, Jingang;Wang, Xian
通讯作者：
Wang, Xian
DOI：
10.1016/j.jamcollsurg.2006.12.029
发表时间：
2007-03-01
影响因子：
5.2
作者：
Andersen, Nicholas D.;Monahan, Thomas S.;LoGerfo, Frank W.
通讯作者：
LoGerfo, Frank W.
影响因子：
3.5
作者：
Hlawaty, Hanna;Juan, Aurhe San;Feldman, Laurent J.
通讯作者：
Feldman, Laurent J.