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

• Published in: PloS one 2014-02, Vol.9 (2), p.e89395-e89395
• Main Authors: Yeo, Jiyoun, Crawford, Erin L, Blomquist, Thomas M, Stanoszek, Lauren M, Dannemiller, Rachel E, Zyrek, Jill, De Las Casas, Luis E, Khuder, Sadik A, Willey, James C
• Format: Article
• Language: English
• Subjects: Amplification; Analysis; Biology; Cancer; Cancer treatment; Color; Competition; Competitive materials; Critical care; Cyclin-dependent kinases; Diagnostic systems; Diagnostic tests; DNA probes; Fitness; Fluorescence; Formaldehyde; Gene expression; Genes; Health sciences; Humans; Hydrolysis; Kinases; Laboratories; Linearity; Lung cancer; Lung diseases; Medical diagnosis; Medical research; Medicine; Methods; Molecular Diagnostic Techniques - methods; Multiplex Polymerase Chain Reaction - methods; Multiplexing; Myc protein; Paraffin; Paraffin Embedding; Pathology; Polymerase chain reaction; Quality assessment; Quality control; Quality management; Quality standards; Reagents; Real time; Real-Time Polymerase Chain Reaction - methods; Reproducibility; Reverse transcription; Ribonucleic acid; RNA; Surgery; Tissue Fixation
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0089395

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. 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. 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 (