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High throughput RNAi assay optimization using adherent cell cytometry.

Nabzdyk CS, Chun M, Pradhan L, Logerfo FW - J Transl Med (2011)

Bottom Line: 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 technology can accelerate in vitro cell assays and thus save costs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Vascular and Endovascular Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

ABSTRACT

Background: 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).

Methods: AoSMC were seeded at a density of 3000-8000 cells/well of a 96 well 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.

Results: 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.

Conclusion: 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.

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Related in: MedlinePlus

Experimental conditions and total number of samples. Human aortic smooth muscle cells were transfected in the presence or absence of a transfection reagent (HiPerfect™ or RNAiMax™; 0.375 μl/100 μl each). Each group was further divided into four conditions: no siRNA, 50 nM unlabeled control siRNA, and 5 nM and 50 nM of siGLO Red transfection indicator. Each treatment was carried out in quadruplicates and each experiment was repeated three times for a total sample size of 144.
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Figure 1: Experimental conditions and total number of samples. Human aortic smooth muscle cells were transfected in the presence or absence of a transfection reagent (HiPerfect™ or RNAiMax™; 0.375 μl/100 μl each). Each group was further divided into four conditions: no siRNA, 50 nM unlabeled control siRNA, and 5 nM and 50 nM of siGLO Red transfection indicator. Each treatment was carried out in quadruplicates and each experiment was repeated three times for a total sample size of 144.

Mentions: Confluent AoSMCs were seeded at a density of 3000-8000 cells/well in a BD Falcon 96-well black-bottom plate (Fisher, Pittsburg, PA). 24 hours later, cells were transfected with either non-targeting unlabeled siRNA (50 nM) (CAT#ID D-001206-13-20, Dharmacon, Lafayette, CO), siRNA targeting human MARCKS (CAT#ID D-004772-04, Dharmacon, Lafayette, CO) or siGLO Red (5 or 50 nM) (Dharmacon, Lafayette, CO) using no transfection reagent, HiPerfect (Qiagen, Valencia, CA), or Lipofectamine RNAi Max (Invitrogen, Carlsbad, CA) as recommended by the manufacturer. A master mix was created for each individual condition in order to eliminate pipetting errors and to increase consistency between each well. The experimental set-up of the twelve conditions for each cell type is outlined in Figure 1.


High throughput RNAi assay optimization using adherent cell cytometry.

Nabzdyk CS, Chun M, Pradhan L, Logerfo FW - J Transl Med (2011)

Experimental conditions and total number of samples. Human aortic smooth muscle cells were transfected in the presence or absence of a transfection reagent (HiPerfect™ or RNAiMax™; 0.375 μl/100 μl each). Each group was further divided into four conditions: no siRNA, 50 nM unlabeled control siRNA, and 5 nM and 50 nM of siGLO Red transfection indicator. Each treatment was carried out in quadruplicates and each experiment was repeated three times for a total sample size of 144.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3111359&req=5

Figure 1: Experimental conditions and total number of samples. Human aortic smooth muscle cells were transfected in the presence or absence of a transfection reagent (HiPerfect™ or RNAiMax™; 0.375 μl/100 μl each). Each group was further divided into four conditions: no siRNA, 50 nM unlabeled control siRNA, and 5 nM and 50 nM of siGLO Red transfection indicator. Each treatment was carried out in quadruplicates and each experiment was repeated three times for a total sample size of 144.
Mentions: Confluent AoSMCs were seeded at a density of 3000-8000 cells/well in a BD Falcon 96-well black-bottom plate (Fisher, Pittsburg, PA). 24 hours later, cells were transfected with either non-targeting unlabeled siRNA (50 nM) (CAT#ID D-001206-13-20, Dharmacon, Lafayette, CO), siRNA targeting human MARCKS (CAT#ID D-004772-04, Dharmacon, Lafayette, CO) or siGLO Red (5 or 50 nM) (Dharmacon, Lafayette, CO) using no transfection reagent, HiPerfect (Qiagen, Valencia, CA), or Lipofectamine RNAi Max (Invitrogen, Carlsbad, CA) as recommended by the manufacturer. A master mix was created for each individual condition in order to eliminate pipetting errors and to increase consistency between each well. The experimental set-up of the twelve conditions for each cell type is outlined in Figure 1.

Bottom Line: 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 technology can accelerate in vitro cell assays and thus save costs.

View Article: PubMed Central - HTML - PubMed

Affiliation: Division of Vascular and Endovascular Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.

ABSTRACT

Background: 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).

Methods: AoSMC were seeded at a density of 3000-8000 cells/well of a 96 well 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.

Results: 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.

Conclusion: 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.

Show MeSH
Related in: MedlinePlus