title: Evaluation of the ability of two transfection reagents to deliver small interfering RNA molecules to equine and guinea pig cartilage in vitro.
abstract: OBJECTIVE
  To evaluate 2 commercially available transfection reagents for transfection efficiency and distribution of small interfering RNA (siRNA) molecules to chondrocytes in monolayer cultures and full-thickness cartilage explants from guinea pigs and horses.
  
  
  METHODS
  Cartilage explants from 5 one-month-old and 3 adult guinea pigs and 5 adult clinically normal horses.
  
  
  METHODS
  Monolayer chondrocytes and uniform cartilage explants were exposed to 1 of 2 siRNA transfection complexes according to manufacturers' protocols (1μM [1×]). Additionally, monolayer chondrocytes were exposed to 2× the suggested amount of a proprietary siRNA molecule. Full-thickness cartilage explants were treated with 1× (1μM), 2× (2μM), and 4× (4μM) or 1× (0.13μM), 4× (0.52μM), and 8× (1.04μM) the recommended concentrations of the proprietary siRNA and the cationic liposome siRNA, respectively, in equivalent media volumes. Use of fluorescent siRNA duplexes allowed quantification of transfected cells via flow cytometry and direct visualization of the depth and distribution of in situ transfection via fluorescent microscopy.
  
  
  RESULTS
  With both transfection reagents, > 90% of monolayer chondrocytes were transfected. In explants, only use of the proprietary molecule achieved > 50% transfection efficiency, whereas use of the cationic liposome achieved < 20%. Only the proprietary molecule-treated cartilage consistently contained fluorescent cells throughout all zones; the cationic liposome-transfected chondrocytes were restricted to explant surfaces.
  
  
  CONCLUSIONS
  Robust transfection of chondrocytes in monolayer was achieved with both reagents, but only use of the proprietary molecule attained effective full-thickness transfection of explants that may allow relevant transcript reduction via RNAi.
authors.affiliation: Willamette Valley Equine Surgical and Medical Center, Aurora, OR 97002, USA.
authors.forename: Sarah S
authors.initials: SS
authors.lastname: Dougherty
authors.forename: Kelly S
authors.initials: KS
authors.lastname: Santangelo
authors.forename: Alicia L
authors.initials: AL
authors.lastname: Bertone
mesh_terms: D000818:Animals; D002356:Cartilage / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D019902:Chondrocytes / Q000166:cytology / Q000378:metabolism* / Q000473:pathology; D005434:Flow Cytometry / Q000662:veterinary; D006168:Guinea Pigs; D006736:Horses; D008297:Male; D008856:Microscopy, Fluorescence / Q000662:veterinary; D010003:Osteoarthritis / Q000378:metabolism* / Q000473:pathology; D034741:RNA, Small Interfering / Q000235:genetics / Q000378:metabolism*; D014162:Transfection / Q000379:methods / Q000662:veterinary*
chemical_list: D034741:RNA, Small Interfering