title: Profound influence of microarray scanner characteristics on gene expression ratios: analysis and procedure for correction.
abstract: BACKGROUND
  High throughput gene expression data from spotted cDNA microarrays are collected by scanning the signal intensities of the corresponding spots by dedicated fluorescence scanners. The major scanner settings for increasing the spot intensities are the laser power and the voltage of the photomultiplier tube (PMT). It is required that the expression ratios are independent of these settings. We have investigated the relationships between PMT voltage, spot intensities, and expression ratios for different scanners, in order to define an optimal scanning procedure.
  
  
  RESULTS
  All scanners showed a limited intensity range from 200 to 50 000 (mean spot intensity), for which the expression ratios were independent of PMT voltage. This usable intensity range was considerably less than the maximum detection range of the PMTs. The use of spot and background intensities outside this range led to errors in the ratios. The errors at high intensities were caused by saturation of pixel intensities within the spots. An algorithm was developed to correct the intensities of these spots, and, hence, extend the upper limit of the usable intensity range.
  
  
  CONCLUSIONS
  It is suggested that the PMT voltage should be increased to avoid intensities of the weakest spots below the usable range, allowing the brightest spots to reach the level of saturation. Subsequently, a second set of images should be acquired with a lower PMT setting such that no pixels are in saturation. Reliable data for spots with saturation in the first set of images can easily be extracted from the second set of images by the use of our algorithm. This procedure would lead to an increase in the accuracy of the data and in the number of data points achieved in each experiment compared to traditional procedures.
authors.affiliation: Department of Biophysics, The Norwegian Radium Hospital, Oslo, Norway. heidi.lyng@labmed.uio.no
authors.forename: Heidi
authors.initials: H
authors.lastname: Lyng
authors.forename: Azadeh
authors.initials: A
authors.lastname: Badiee
authors.forename: Debbie H
authors.initials: DH
authors.lastname: Svendsrud
authors.forename: Eivind
authors.initials: E
authors.lastname: Hovig
authors.forename: Ola
authors.initials: O
authors.lastname: Myklebost
authors.forename: Trond
authors.initials: T
authors.lastname: Stokke
mesh_terms: D000465:Algorithms; D002232:Carbocyanines / Q000737:chemistry; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D005260:Female; D020869:Gene Expression Profiling / Q000295:instrumentation / Q000379:methods / Q000592:standards*; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods / Q000592:standards*; D015203:Reproducibility of Results; D002583:Uterine Cervical Neoplasms / Q000235:genetics
chemical_list: D002232:Carbocyanines; D018076:DNA, Complementary; C089187:cyanine dye 3; C085321:cyanine dye 5