title: Development of a novel ozone- and photo-stable HyPer5 red fluorescent dye for array CGH and microarray gene expression analysis with consistent performance irrespective of environmental conditions.
abstract: BACKGROUND
  Array-based comparative genomic hybridization (CGH) and gene expression profiling have become vital techniques for identifying molecular defects underlying genetic diseases. Regardless of the microarray platform, cyanine dyes (Cy3 and Cy5) are one of the most widely used fluorescent dye pairs for microarray analysis owing to their brightness and ease of incorporation, enabling high level of assay sensitivity. However, combining both dyes on arrays can become problematic during summer months when ozone levels rise to near 25 parts per billion (ppb). Under such conditions, Cy5 is known to rapidly degrade leading to loss of signal from either "homebrew" or commercial arrays. Cy5 can also suffer disproportionately from dye photobleaching resulting in distortion of (Cy5/Cy3) ratios used in copy number analysis. Our laboratory has been active in fluorescent dye research to find a suitable alternative to Cy5 that is stable to ozone and resistant to photo-bleaching. Here, we report on the development of such a dye, called HyPer5, and describe its' exceptional ozone and photostable properties on microarrays.
  
  
  RESULTS
  Our results show HyPer5 signal to be stable to high ozone levels. Repeated exposure of mouse arrays hybridized with HyPer5-labeled cDNA to 300 ppb ozone at 5, 10 and 15 minute intervals resulted in no signal loss from the dye. In comparison, Cy5 arrays showed a dramatic 80% decrease in total signal during the same interval. Photobleaching experiments show HyPer5 to be resistant to light induced damage with 3- fold improvement in dye stability over Cy5. In high resolution array CGH experiments, HyPer5 is demonstrated to detect chromosomal aberrations at loci 2p21-16.3 and 15q26.3-26.2 from three patient sample using bacterial artificial chromosome (BAC) arrays. The photostability of HyPer5 is further documented by repeat array scanning without loss of detection. Additionally, HyPer5 arrays are shown to preserve sensitivity and data quality from gene expression experiments.
  
  
  CONCLUSIONS
  HyPer5 is a red fluorescent dye that behaves functionally similar to Cy5 except in stability to ozone and light. HyPer5 is demonstrated to be resistant to ozone at up to 300 ppb, levels significantly higher than commonly observed during summer months. Consequently, HyPer5 dye can be used in parallel with Cy3 under any environmental conditions in array experiments.
authors.affiliation: GE Healthcare, 800 Centennial Ave, Piscataway, NJ 08855, USA. mubasher.dar@ge.com
authors.forename: Mubasher
authors.initials: M
authors.lastname: Dar
authors.forename: Theresa
authors.initials: T
authors.lastname: Giesler
authors.forename: Rob
authors.initials: R
authors.lastname: Richardson
authors.forename: Christine
authors.initials: C
authors.lastname: Cai
authors.forename: Mike
authors.initials: M
authors.lastname: Cooper
authors.forename: Shahin
authors.initials: S
authors.lastname: Lavasani
authors.forename: Peter
authors.initials: P
authors.lastname: Kille
authors.forename: Thierry
authors.initials: T
authors.lastname: Voet
authors.forename: Joris
authors.initials: J
authors.lastname: Vermeesch
mesh_terms: D002232:Carbocyanines / Q000737:chemistry* / Q000528:radiation effects; D055028:Comparative Genomic Hybridization / Q000379:methods*; D018076:DNA, Complementary; D005456:Fluorescent Dyes / Q000737:chemistry* / Q000528:radiation effects; D020869:Gene Expression Profiling / Q000379:methods; D008027:Light; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D010126:Ozone / Q000737:chemistry*; D038761:Photobleaching; D012680:Sensitivity and Specificity
chemical_list: D002232:Carbocyanines; D018076:DNA, Complementary; D005456:Fluorescent Dyes; D010126:Ozone