title: A multiplex two-color real-time PCR method for quality-controlled molecular diagnostic testing of FFPE samples.
abstract: BACKGROUND
  Reverse transcription quantitative real-time PCR (RT-qPCR) tests support personalized cancer treatment through more clinically meaningful diagnosis. However, samples obtained through standard clinical pathology procedures are formalin-fixed, paraffin-embedded (FFPE) and yield small samples with low integrity RNA containing PCR interfering substances. RT-qPCR tests able to assess FFPE samples with quality control and inter-laboratory reproducibility are needed.
  
  
  METHODS
  We developed an RT-qPCR method by which 1) each gene was measured relative to a known number of its respective competitive internal standard molecules to control for interfering substances, 2) two-color fluorometric hydrolysis probes enabled analysis on a real-time platform, 3) external standards controlled for variation in probe fluorescence intensity, and 4) pre-amplification maximized signal from FFPE RNA samples. Reagents were developed for four genes comprised by a previously reported lung cancer diagnostic test (LCDT) then subjected to analytical validation using synthetic native templates as test articles to assess linearity, signal-to-analyte response, lower detection threshold, imprecision and accuracy. Fitness of this method and these reagents for clinical testing was assessed in FFPE normal (N = 10) and malignant (N = 10) lung samples.
  
  
  RESULTS
  Reagents for each of four genes, MYC, E2F1, CDKN1A and ACTB comprised by the LCDT had acceptable linearity (R(2)>0.99), signal-to-analyte response (slope 1.0 ± 0.05), lower detection threshold (<10 molecules) and imprecision (CV <20%). Poisson analysis confirmed accuracy of internal standard concentrations. Internal standards controlled for experimentally introduced interference, prevented false-negatives and enabled pre-amplification to increase signal without altering measured values. In the fitness for purpose testing of this two-color fluorometric LCDT using surgical FFPE samples, the diagnostic accuracy was 93% which was similar to that previously reported for analysis of fresh samples.
  
  
  CONCLUSIONS
  This quality-controlled two-color fluorometric RT-qPCR approach will facilitate the development of reliable, robust RT-qPCR-based molecular diagnostic tests in FFPE clinical samples.
authors.affiliation: Division of Pulmonary/Critical Care and Sleep Medicine, Department of Medicine, University of Toledo Health Sciences Campus, Toledo, Ohio, United States of America.
authors.forename: Jiyoun
authors.initials: J
authors.lastname: Yeo
authors.affiliation: Division of Pulmonary/Critical Care and Sleep Medicine, Department of Medicine, University of Toledo Health Sciences Campus, Toledo, Ohio, United States of America.
authors.forename: Erin L
authors.initials: EL
authors.lastname: Crawford
authors.affiliation: Division of Pulmonary/Critical Care and Sleep Medicine, Department of Medicine, University of Toledo Health Sciences Campus, Toledo, Ohio, United States of America.
authors.forename: Thomas M
authors.initials: TM
authors.lastname: Blomquist
authors.affiliation: Division of Pulmonary/Critical Care and Sleep Medicine, Department of Medicine, University of Toledo Health Sciences Campus, Toledo, Ohio, United States of America.
authors.forename: Lauren M
authors.initials: LM
authors.lastname: Stanoszek
authors.affiliation: Division of Pulmonary/Critical Care and Sleep Medicine, Department of Medicine, University of Toledo Health Sciences Campus, Toledo, Ohio, United States of America.
authors.forename: Rachel E
authors.initials: RE
authors.lastname: Dannemiller
authors.affiliation: Department of Pathology, University of Toledo Health Sciences Campus, Toledo, Ohio, United States of America.
authors.forename: Jill
authors.initials: J
authors.lastname: Zyrek
authors.affiliation: Department of Pathology, University of Toledo Health Sciences Campus, Toledo, Ohio, United States of America.
authors.forename: Luis E
authors.initials: LE
authors.lastname: De Las Casas
authors.affiliation: Division of Pulmonary/Critical Care and Sleep Medicine, Department of Medicine, University of Toledo Health Sciences Campus, Toledo, Ohio, United States of America.
authors.forename: Sadik A
authors.initials: SA
authors.lastname: Khuder
authors.affiliation: Division of Pulmonary/Critical Care and Sleep Medicine, Department of Medicine, University of Toledo Health Sciences Campus, Toledo, Ohio, United States of America ; Department of Pathology, University of Toledo Health Sciences Campus, Toledo, Ohio, United States of America.
authors.forename: James C
authors.initials: JC
authors.lastname: Willey
mesh_terms: D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D016612:Paraffin Embedding; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D016707:Tissue Fixation