Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes

The detection and quantification of leukemia-associated fusion gene transcripts play important roles in the diagnosis and follow-up of leukemias. To establish a standardized method without interlaboratory discrepancies, we developed a novel one-step reverse transcription quantitative PCR (RT-qPCR) a...

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Bibliographic Details
Published in:PloS one 2018-10, Vol.13 (10), p.e0202429-e0202429
Main Authors: Tsuchiya, Koji, Tabe, Yoko, Ai, Tomohiko, Ohkawa, Takahiro, Usui, Kengo, Yuri, Maiko, Misawa, Shigeki, Morishita, Soji, Takaku, Tomoiku, Kakimoto, Atsushi, Yang, Haeun, Matsushita, Hiromichi, Hanami, Takeshi, Yamanaka, Yasunari, Okuzawa, Atsushi, Horii, Takashi, Hayashizaki, Yoshihide, Ohsaka, Akimichi
Format: Article
Language:English
Subjects:
Analysis
Assaying
Biology and Life Sciences
Cancer
Deoxyribonucleic acid
DNA
Feasibility studies
Fluorescence
Fusion protein
Fusion proteins
Gene expression
Genes
Genetic aspects
Genetic testing
Genomics
Hematology
Hospitals
Hybridization
Innovations
Isoforms
Laboratories
Leukemia
Life sciences
Medical diagnosis
Medicine and Health Sciences
Melting
Melting curve
Minimal residual disease
Oligonucleotides
Physical Sciences
Polymerase chain reaction
Preventive medicine
Quality control
Research and Analysis Methods
Reverse transcription
Runx1 protein
Stem cells
Systems development
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Description
Summary:The detection and quantification of leukemia-associated fusion gene transcripts play important roles in the diagnosis and follow-up of leukemias. To establish a standardized method without interlaboratory discrepancies, we developed a novel one-step reverse transcription quantitative PCR (RT-qPCR) assay, called "the Eprobe leukemia assay," for major and minor BCR-ABL1, RUNX1-RUNX1T1, and various isoforms of PML-RARA. This assay is comprised of Eprobes that are exciton-controlled hybridization-sensitive fluorescent oligonucleotides. Melting curve analyses were performed on synthetic quantitative standard RNAs with strict quality control. Quantification capacity was evaluated by comparison with TaqMan RT-qPCR using 67 primary leukemia patient samples. The lower limit of detection and the limit of quantification of this assay were less than 31.3 copies/reaction and 62.5 copies/reaction, respectively. This assay correctly detected the fusion genes in samples with 100% sensitivity and specificity. The specificity of the reactions was confirmed by melting curve analyses. The assay detected low-level expression of minor BCR-ABL1 co-expressed with major BCR-ABL1. These results illustrate the feasibility and high accuracy of the Eprobe leukemia assay, even for minimal residual disease monitoring.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0202429