A multiplex two-color real-time PCR method for quality-controlled molecular diagnostic testing of FFPE samples

Jiyoun Yeo, Erin L. Crawford, Thomas M. Blomquist, Lauren M. Stanoszek, Rachel E. Dannemiller, Jill Zyrek, Luis E. De Las Casas, Sadik A. Khuder, James C. Willey

Research output: Contribution to journalArticlepeer-review

8 Scopus citations


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 (R2>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.

Original languageEnglish (US)
Article numbere89395
JournalPloS one
Issue number2
StatePublished - Feb 21 2014
Externally publishedYes

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)
  • General


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