title: Temperature-controlled primer limit for multiplexing of rapid, quantitative reverse transcription-PCR assays: application to intraoperative cancer diagnostics.
abstract: BACKGROUND
  Rapid-cycling, real-time PCR instruments bring the opportunity for improved intraoperative detection of metastasis to sentinel lymph nodes. Rapid, standardized, and internally controlled assays need to be developed that are sensitive and accurate.
  
  
  METHODS
  We describe rapid, multiplexed, internally controlled, quantitative reverse transcription-PCR (QRT-PCR) assays for tyrosinase and carcinoembryonic antigen mRNAs on the SmartCycler (Cepheid). We used a temperature-controlled primer-limiting approach to eliminate amplification of the endogenous control gene as soon as its signal had reached threshold. Positive-control oligonucleotide mimics were incorporated into all reactions to differentiate failed reactions from true negative samples.
  
  
  RESULTS
  The optimized assays for rapid QRT-PCR yielded results with threshold cycle values that were only 1-2 cycles higher than slower, more conventional protocols. In rapid PCR, the temperature-controlled multiplex assay was quantitative over a dynamic range of at least 15 cycles, compared with only 6 cycles for conventional multiplexing methods. All histologically positive lymph nodes examined were also QRT-PCR positive for the appropriate marker, and the exogenous, internal positive-control mimics produced signals in all negative samples.
  
  
  CONCLUSIONS
  Internally controlled, rapid QRT-PCR assays can be performed in an intraoperative time frame and with sufficient sensitivity to detect histologically identified metastases to lymph nodes.
authors.affiliation: Division of Thoracic Surgery, University of Pittsburgh Medical Center, PA 15213, USA.
authors.forename: Siva
authors.initials: S
authors.lastname: Raja
authors.forename: Talal
authors.initials: T
authors.lastname: El-Hefnawy
authors.forename: Lori A
authors.initials: LA
authors.lastname: Kelly
authors.forename: Melissa L
authors.initials: ML
authors.lastname: Chestney
authors.forename: James D
authors.initials: JD
authors.lastname: Luketich
authors.forename: Tony E
authors.initials: TE
authors.lastname: Godfrey
mesh_terms: D001483:Base Sequence; D002272:Carcinoembryonic Antigen / Q000235:genetics; D017931:DNA Primers; D004938:Esophageal Neoplasms / Q000473:pathology; D005260:Female; D006801:Humans; D007432:Intraoperative Period; D008198:Lymph Nodes / Q000473:pathology*; D008207:Lymphatic Metastasis / Q000175:diagnosis*; D008545:Melanoma / Q000473:pathology; D008969:Molecular Sequence Data; D014442:Monophenol Monooxygenase / Q000235:genetics; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013696:Temperature
chemical_list: D002272:Carcinoembryonic Antigen; D017931:DNA Primers; D012333:RNA, Messenger; D014442:Monophenol Monooxygenase