title: High throughput RNAi assay optimization using adherent cell cytometry.
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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Division of Vascular and Endovascular Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA.
authors.forename: Christoph S
authors.initials: CS
authors.lastname: Nabzdyk
authors.forename: Maggie
authors.initials: M
authors.lastname: Chun
authors.forename: Leena
authors.initials: L
authors.lastname: Pradhan
authors.forename: Frank W
authors.initials: FW
authors.lastname: Logerfo
mesh_terms: D001011:Aorta / Q000166:cytology; D002448:Cell Adhesion; D002452:Cell Count; D016923:Cell Death; D002478:Cells, Cultured; D005434:Flow Cytometry / Q000379:methods*; D005456:Fluorescent Dyes / Q000378:metabolism; D057166:High-Throughput Screening Assays / Q000379:methods*; D006801:Humans; D007202:Indicators and Reagents; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000378:metabolism; D032389:Myocytes, Smooth Muscle / Q000166:cytology* / Q000378:metabolism*; D000076250:Myristoylated Alanine-Rich C Kinase Substrate; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D014162:Transfection
chemical_list: D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D047908:Intracellular Signaling Peptides and Proteins; C000619710:MARCKS protein, human; D008565:Membrane Proteins; D034741:RNA, Small Interfering; D000076250:Myristoylated Alanine-Rich C Kinase Substrate