title: Quantitative real-time reverse transcription-PCR assay for urokinase plasminogen activator, plasminogen activator inhibitor type 1, and tissue metalloproteinase inhibitor type 1 gene expressions in primary breast cancer.
abstract: BACKGROUND
  The plasminogen activation system and matrix metalloproteinases (MMPs) play a key role in the degradation of basement membrane and extracellular matrix in tissue remodeling, cancer cell invasion, and metastasis.
  
  
  METHODS
  Quantitative real-time reverse-transcription-PCR (RT-PCR) assays were developed to quantify urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor type 1 (PAI-1), and tissue metalloproteinase inhibitor type 1 (TIMP-1) mRNA in 54 breast cancer tissues. Gene fragments were amplified in a LightCycler real-time PCR system using gene-specific primers and SYBR Green I. The results were normalized to beta-actin mRNA. We also quantified antigen and functional concentrations of these components.
  
  
  RESULTS
  The intra- and interassay variabilities for mRNA quantification showed mean SDs for the crossing point of 0.12 and 0.15 cycles, respectively. PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and PAI-1 and uPA functional concentrations increased with tumor severity; the increase was statistically significant for PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations and for uPA functional concentrations. Node-positive patients showed significantly higher PAI-1, uPA, and TIMP-1 mRNA and antigen concentrations than those who were node negative.
  
  
  CONCLUSIONS
  Quantitative real-time RT-PCR is a highly sensitive, reproducible, and fast method for measuring gene expression of PAI-1, uPA, and TIMP-1 in breast cancer. These components may be involved in breast cancer development, and increased mRNA expression may be associated with a worse prognosis.
authors.affiliation: Hospital Universitario La Fe, Centro de Investigación, Avda. Campanar 21, 46009 Valencia, Spain.
authors.forename: Remedios
authors.initials: R
authors.lastname: Castelló
authors.forename: Amparo
authors.initials: A
authors.lastname: Estellés
authors.forename: Carlos
authors.initials: C
authors.lastname: Vázquez
authors.forename: Cristina
authors.initials: C
authors.lastname: Falcó
authors.forename: Francisco
authors.initials: F
authors.lastname: España
authors.forename: Sergio M
authors.initials: SM
authors.lastname: Almenar
authors.forename: Carlos
authors.initials: C
authors.lastname: Fuster
authors.forename: Justo
authors.initials: J
authors.lastname: Aznar
mesh_terms: D000328:Adult; D000368:Aged; D001940:Breast / Q000378:metabolism; D001943:Breast Neoplasms / Q000378:metabolism* / Q000473:pathology; D005260:Female; D006801:Humans; D008875:Middle Aged; D017395:Plasminogen Activator Inhibitor 1 / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D012720:Severity of Illness Index; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism*; D014568:Urokinase-Type Plasminogen Activator / Q000235:genetics / Q000378:metabolism*
chemical_list: D017395:Plasminogen Activator Inhibitor 1; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D014568:Urokinase-Type Plasminogen Activator