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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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Comparison of transfection reagents in siGLO Red transfected AoSMCs. (A, D, and G) No transfection reagent with 50 nM control siRNA, 5 nM siGLO Red and 50 nM siGLO Red respectively. (B, E, and H) HiPerfect transfection reagent (0.375 μl/100 μl) with 50 nM control siRNA, 5 nM siGLO Red and 50 nM siGLO Red respectively. (C, F, and I) RNAiMax transfection reagent (0.375 μl/100 μl) with 50 nM control siRNA, 5 nM siGLO Red and 50 nM siGLO Red respectively. (J) Representativepicture of 50 nM siGLO Red + RNAiMax transfected AoSMCs used for quantification. AoSMCs were also stained with Hoechst nuclei stain. Both fluorescent signals were confined within the cells. (K) Represents Figure 4J with the gated fluorescent events (red dots/circles = siGLO Red; blue circles = Hoechst stained nuclei). Note the exact overlay of actual and detected fluorescent events. The vast majority of gated signals originated from the AoSMCs.
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Figure 4: Comparison of transfection reagents in siGLO Red transfected AoSMCs. (A, D, and G) No transfection reagent with 50 nM control siRNA, 5 nM siGLO Red and 50 nM siGLO Red respectively. (B, E, and H) HiPerfect transfection reagent (0.375 μl/100 μl) with 50 nM control siRNA, 5 nM siGLO Red and 50 nM siGLO Red respectively. (C, F, and I) RNAiMax transfection reagent (0.375 μl/100 μl) with 50 nM control siRNA, 5 nM siGLO Red and 50 nM siGLO Red respectively. (J) Representativepicture of 50 nM siGLO Red + RNAiMax transfected AoSMCs used for quantification. AoSMCs were also stained with Hoechst nuclei stain. Both fluorescent signals were confined within the cells. (K) Represents Figure 4J with the gated fluorescent events (red dots/circles = siGLO Red; blue circles = Hoechst stained nuclei). Note the exact overlay of actual and detected fluorescent events. The vast majority of gated signals originated from the AoSMCs.

Mentions: Visualization of the transfected cells confirmed that the vast majority of red fluorescence originated from siGLO Red transfected AoSMCs with only minimal extracellular signals (Figure 4A-I). In addition, the adherent cell cytometer precisely detected the nuclei and fluorescently labeled siGLO Red siRNA within the AoSMCs (Figure 4J and 4K).


High throughput RNAi assay optimization using adherent cell cytometry.

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

Comparison of transfection reagents in siGLO Red transfected AoSMCs. (A, D, and G) No transfection reagent with 50 nM control siRNA, 5 nM siGLO Red and 50 nM siGLO Red respectively. (B, E, and H) HiPerfect transfection reagent (0.375 μl/100 μl) with 50 nM control siRNA, 5 nM siGLO Red and 50 nM siGLO Red respectively. (C, F, and I) RNAiMax transfection reagent (0.375 μl/100 μl) with 50 nM control siRNA, 5 nM siGLO Red and 50 nM siGLO Red respectively. (J) Representativepicture of 50 nM siGLO Red + RNAiMax transfected AoSMCs used for quantification. AoSMCs were also stained with Hoechst nuclei stain. Both fluorescent signals were confined within the cells. (K) Represents Figure 4J with the gated fluorescent events (red dots/circles = siGLO Red; blue circles = Hoechst stained nuclei). Note the exact overlay of actual and detected fluorescent events. The vast majority of gated signals originated from the AoSMCs.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 4: Comparison of transfection reagents in siGLO Red transfected AoSMCs. (A, D, and G) No transfection reagent with 50 nM control siRNA, 5 nM siGLO Red and 50 nM siGLO Red respectively. (B, E, and H) HiPerfect transfection reagent (0.375 μl/100 μl) with 50 nM control siRNA, 5 nM siGLO Red and 50 nM siGLO Red respectively. (C, F, and I) RNAiMax transfection reagent (0.375 μl/100 μl) with 50 nM control siRNA, 5 nM siGLO Red and 50 nM siGLO Red respectively. (J) Representativepicture of 50 nM siGLO Red + RNAiMax transfected AoSMCs used for quantification. AoSMCs were also stained with Hoechst nuclei stain. Both fluorescent signals were confined within the cells. (K) Represents Figure 4J with the gated fluorescent events (red dots/circles = siGLO Red; blue circles = Hoechst stained nuclei). Note the exact overlay of actual and detected fluorescent events. The vast majority of gated signals originated from the AoSMCs.
Mentions: Visualization of the transfected cells confirmed that the vast majority of red fluorescence originated from siGLO Red transfected AoSMCs with only minimal extracellular signals (Figure 4A-I). In addition, the adherent cell cytometer precisely detected the nuclei and fluorescently labeled siGLO Red siRNA within the AoSMCs (Figure 4J and 4K).

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