title: Rapid selection of differentially expressed genes in TNF[alpha]-activated endothelial cells.
abstract: BACKGROUND
  RNA differential display (DD) RT-PCR is a useful method to identify and clone differentially expressed genes. However, the rate of false positives and redundancy associated with this PCR-based method as well as laborious downstream screening steps constitute major limitations. Here we present DD RT-PCR and reverse northern (RN) protocols allowing rapid and acurate identification of genes upregulated in porcine endothelial cells (EC) in response to TNFalpha.
  
  
  METHODS
  The housekeeping gene beta-actin was used to investigate mispriming and to set up optimal conditions for DD-RT-PCR and RN. In this study DD was performed to compare resting and TNFalpha-activated ECs. Selection of DD-fragments was performed following 30-cycles of PCR using serial dilutions of template cDNA and regulation of 6 out of 17 candidates genes were first confirmed by semi-quantitative RN.
  
  
  RESULTS
  Using this protocol, 5 out of 6 DD-fragments were further confirmed to be upregulated by Northern blot, and 3 novel porcine cDNAs were cloned including the pro-apoptotic member of the Bcl-2 family, Noxa.
  
  
  CONCLUSIONS
  In this study we demonstrate that the combination of DD-RT-PCR and RN, which efficiently reduces the number of false positive candidates derived from mispriming at the screening step, allows a rapid identification of differentially expressed genes.
authors.affiliation: Institut National de la Recherche Médicale and Institut de Transplantation et de Recherche en Transplantation, Nantes, FR.
authors.forename: Takaharu
authors.initials: T
authors.lastname: Nagasaka
authors.forename: Gwénola
authors.initials: G
authors.lastname: Boulday
authors.forename: Christopher C
authors.initials: CC
authors.lastname: Fraser
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Coupel
authors.forename: Flora
authors.initials: F
authors.lastname: Coulon
authors.forename: Laurent
authors.initials: L
authors.lastname: Tesson
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Heslan
authors.forename: Jean-Paul
authors.initials: JP
authors.lastname: Soulillou
authors.forename: Béatrice
authors.initials: B
authors.lastname: Charreau
mesh_terms: D000818:Animals; D001011:Aorta / Q000378:metabolism; D001483:Base Sequence; D015152:Blotting, Northern; D004730:Endothelium, Vascular / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D008969:Molecular Sequence Data; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D013552:Swine; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*; D015854:Up-Regulation
chemical_list: D019253:Proto-Oncogene Proteins c-bcl-2; D014409:Tumor Necrosis Factor-alpha