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

Koji Tsuchiya, Yoko Tabe, Tomohiko Ai, Takahiro Ohkawa, Kengo Usui, Maiko Yuri, Shigeki Misawa, Soji Morishita, Tomoiku Takaku, Atsushi Kakimoto, Haeun Yang, Hiromichi Matsushita, Takeshi Hanami, Yasunari Yamanaka, Atsushi Okuzawa, Takashi Horii, Yoshihide Hayashizaki, Akimichi Ohsaka

Research output: Contribution to journalArticle

1 Scopus citations

Abstract

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.

Original languageEnglish (US)
Article numbere0202429
JournalPLoS One
Volume13
Issue number10
DOIs
StatePublished - Oct 1 2018
Externally publishedYes

ASJC Scopus subject areas

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

Fingerprint Dive into the research topics of 'Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes'. Together they form a unique fingerprint.

  • Cite this

    Tsuchiya, K., Tabe, Y., Ai, T., Ohkawa, T., Usui, K., Yuri, M., Misawa, S., Morishita, S., Takaku, T., Kakimoto, A., Yang, H., Matsushita, H., Hanami, T., Yamanaka, Y., Okuzawa, A., Horii, T., Hayashizaki, Y., & Ohsaka, A. (2018). Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes. PLoS One, 13(10), [e0202429]. https://doi.org/10.1371/journal.pone.0202429