title: Comparing gene discovery from Affymetrix GeneChip microarrays and Clontech PCR-select cDNA subtraction: a case study.
abstract: BACKGROUND
  Several high throughput technologies have been employed to identify differentially regulated genes that may be molecular targets for drug discovery. Here we compared the sets of differentially regulated genes discovered using two experimental approaches: a subtracted suppressive hybridization (SSH) cDNA library methodology and Affymetrix GeneChip technology. In this "case study" we explored the transcriptional pattern changes during the in vitro differentiation of human monocytes to myeloid dendritic cells (DC), and evaluated the potential for novel gene discovery using the SSH methodology.
  
  
  RESULTS
  The same RNA samples isolated from peripheral blood monocyte precursors and immature DC (iDC) were used for GeneChip microarray probing and SSH cDNA library construction. 10,000 clones from each of the two-way SSH libraries (iDC-monocytes and monocytes-iDC) were picked for sequencing. About 2000 transcripts were identified for each library from 8000 successful sequences. Only 70% to 75% of these transcripts were represented on the U95 series GeneChip microarrays, implying that 25% to 30% of these transcripts might not have been identified in a study based only on GeneChip microarrays. In addition, about 10% of these transcripts appeared to be "novel", although these have not yet been closely examined. Among the transcripts that are also represented on the chips, about a third were concordantly discovered as differentially regulated between iDC and monocytes by GeneChip microarray transcript profiling. The remaining two thirds were either not inferred as differentially regulated from GeneChip microarray data, or were called differentially regulated but in the opposite direction. This underscores the importance both of generating reciprocal pairs of SSH libraries, and of real-time RT-PCR confirmation of the results.
  
  
  CONCLUSIONS
  This study suggests that SSH could be used as an alternative and complementary transcript profiling tool to GeneChip microarrays, especially in identifying novel genes and transcripts of low abundance.
authors.affiliation: Aventis Pharmaceuticals, Bridgewater, NJ 08887, USA. wuxiong.cao@aventis.com
authors.forename: Wuxiong
authors.initials: W
authors.lastname: Cao
authors.forename: Charles
authors.initials: C
authors.lastname: Epstein
authors.forename: Hong
authors.initials: H
authors.lastname: Liu
authors.forename: Craig
authors.initials: C
authors.lastname: DeLoughery
authors.forename: Nanxiang
authors.initials: N
authors.lastname: Ge
authors.forename: Jieyi
authors.initials: J
authors.lastname: Lin
authors.forename: Rong
authors.initials: R
authors.lastname: Diao
authors.forename: Hui
authors.initials: H
authors.lastname: Cao
authors.forename: Fan
authors.initials: F
authors.lastname: Long
authors.forename: Xin
authors.initials: X
authors.lastname: Zhang
authors.forename: Yangde
authors.initials: Y
authors.lastname: Chen
authors.forename: Paul S
authors.initials: PS
authors.lastname: Wright
authors.forename: Steve
authors.initials: S
authors.lastname: Busch
authors.forename: Michelle
authors.initials: M
authors.lastname: Wenck
authors.forename: Karen
authors.initials: K
authors.lastname: Wong
authors.forename: Alan G
authors.initials: AG
authors.lastname: Saltzman
authors.forename: Zhihua
authors.initials: Z
authors.lastname: Tang
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.forename: Asher
authors.initials: A
authors.lastname: Zilberstein
mesh_terms: D003713:Dendritic Cells / Q000378:metabolism*; D020869:Gene Expression Profiling / Q000379:methods*; D015723:Gene Library; D006801:Humans; D009000:Monocytes / Q000378:metabolism*; D009693:Nucleic Acid Hybridization / Q000379:methods; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000235:genetics / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*
chemical_list: D012313:RNA