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

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

Fingerprint

reverse transcription
gene fusion
Gene Fusion
Transcription
leukemia
Reverse Transcription
Assays
Leukemia
quantitative polymerase chain reaction
Fusion reactions
Genes
Polymerase Chain Reaction
assays
Freezing
melting
Melting
Residual Neoplasm
Oligonucleotides
Quality Control
Limit of Detection

ASJC Scopus subject areas

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

Cite this

Tsuchiya, K., Tabe, Y., Ai, T., Ohkawa, T., Usui, K., Yuri, M., ... 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

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

In: PLoS One, Vol. 13, No. 10, e0202429, 01.10.2018.

Research output: Contribution to journalArticle

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, vol. 13, no. 10, e0202429. https://doi.org/10.1371/journal.pone.0202429
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. / Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes. In: PLoS One. 2018 ; Vol. 13, No. 10.
@article{267c6000d5874fa8ab9410f5faf146e7,
title = "Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes",
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.",
author = "Koji Tsuchiya and Yoko Tabe and Tomohiko Ai and Takahiro Ohkawa and Kengo Usui and Maiko Yuri and Shigeki Misawa and Soji Morishita and Tomoiku Takaku and Atsushi Kakimoto and Haeun Yang and Hiromichi Matsushita and Takeshi Hanami and Yasunari Yamanaka and Atsushi Okuzawa and Takashi Horii and Yoshihide Hayashizaki and Akimichi Ohsaka",
year = "2018",
month = "10",
day = "1",
doi = "10.1371/journal.pone.0202429",
language = "English (US)",
volume = "13",
journal = "PLoS One",
issn = "1932-6203",
publisher = "Public Library of Science",
number = "10",

}

TY - JOUR

T1 - Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes

AU - Tsuchiya, Koji

AU - Tabe, Yoko

AU - Ai, Tomohiko

AU - Ohkawa, Takahiro

AU - Usui, Kengo

AU - Yuri, Maiko

AU - Misawa, Shigeki

AU - Morishita, Soji

AU - Takaku, Tomoiku

AU - Kakimoto, Atsushi

AU - Yang, Haeun

AU - Matsushita, Hiromichi

AU - Hanami, Takeshi

AU - Yamanaka, Yasunari

AU - Okuzawa, Atsushi

AU - Horii, Takashi

AU - Hayashizaki, Yoshihide

AU - Ohsaka, Akimichi

PY - 2018/10/1

Y1 - 2018/10/1

N2 - 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.

AB - 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.

UR - http://www.scopus.com/inward/record.url?scp=85054426804&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85054426804&partnerID=8YFLogxK

U2 - 10.1371/journal.pone.0202429

DO - 10.1371/journal.pone.0202429

M3 - Article

VL - 13

JO - PLoS One

JF - PLoS One

SN - 1932-6203

IS - 10

M1 - e0202429

ER -