title: Real-time telomeric repeat amplification protocol using the duplex scorpion and two reverse primers system: the high sensitive and accurate method for quantification of telomerase activity.
abstract: BACKGROUND
  Real-time quantitative TRAP assays for detection of telomerase activity have been recently developed to eliminate complex post-PCR procedures. However, all of them use the conventional TRAP assay that possesses an unpredictable cascade of events in PCR amplification caused by stagger annealing, which may affect the accuracy of quantitation.
  
  
  METHODS
  A novel RTQ-TRAP method was developed by combining the duplex scorpion with modified TP-TRAP assay that has high fidelity PCR amplification of the telomerase product (DS/TP-TRAP). The synthesized oligonucleotide that represents telomerase products is used to set up a standard curve.
  
  
  RESULTS
  The DS/TP-TRAP method gives the standard curve a dynamic range of 6 orders of magnitude (R(2)=0.9992). It optimizes PCR amplification efficiency and determines telomerase activity in a lower threshold cycle number (Ct value). The method is both accurate and reproducible to measure telomerase activity in human tumor cell lines, and linearity from 1 to 1000 cells could be obtained (R(2)=0.9926). For tumor samples, the results determined by the DS/TP-TRAP assay are comparable to the data obtained with the conventional TRAP method.
  
  
  CONCLUSIONS
  The DS/TP-TRAP assay provides a high sensitive and accurate method for real-time quantitative detection of telomerase activity. It is thus a potential robust tool for application in cancer molecular diagnostics.
authors.affiliation: Tianjin Life Science Research Center, Tianjin Medical University, Tianjin 300070, PR China.
authors.forename: Yan-Ping
authors.initials: YP
authors.lastname: Huang
authors.forename: Zhao-Sheng
authors.initials: ZS
authors.lastname: Liu
authors.forename: Hua
authors.initials: H
authors.lastname: Tang
authors.forename: Min
authors.initials: M
authors.lastname: Liu
authors.forename: Xin
authors.initials: X
authors.lastname: Li
mesh_terms: D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D004797:Enzyme-Linked Immunosorbent Assay; D006801:Humans; D016133:Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D019098:Telomerase / Q000378:metabolism; D016615:Telomere
chemical_list: D017931:DNA Primers; D019098:Telomerase