Pre-clinical validation of a novel, highly sensitive assay to detect PML-RARα mRNA using real-time reverse-transcription polymerase chain reaction

James L. Slack, Wan Li Bi, Kenneth J. Livak, Nike Beaubier, Min Yu, Michelle Clark, Soon H. Kim, Robert E. Gallagher, Cheryl L. Willman

Research output: Contribution to journalArticle

41 Scopus citations

Abstract

We have developed a sensitive and quantitative reverse-transcription polymerase chain reaction (RT-PCR) assay for detection of PML-RARα, the fusion oncogene present as a specific marker in >99% of cases of acute promyelocytic leukemia (APL). The assay is linear over at least 5 orders of magnitude of input DNA or RNA, and detects as few as 4 copies of PML-RARα plasmid DNA. PML-RARα transcripts could be detected in mixtures containing 2 to 5 pg of RNA from fusion-containing cells in a background of 1 μg of RNA from PML-RARα-negative cells. Using 1.0 to 2.5 μg of input RNA, the sensitivity of the assay was between 10-5 and 10-6. Furthermore, determination of GAPDH copy number in each reaction allowed an accurate assessment of sample-to-sample variation in RNA quality and reaction efficiency, with consequent definition of a detection limit for each sample assayed. Using an internal calibrator, assay precision was high, with coefficients of variation between 10 and 20%. An interlaboratory study using coded samples demonstrated excellent reproducibility and high concordance between laboratories. This assay will be used to test the hypothesis that sensitive and quantitative measurement of leukemic burden, during or after therapy of APL, can stratify patients into discrete risk groups, and thereby serve as a basis for risk-adapted therapy in APL.

Original languageEnglish (US)
Pages (from-to)141-149
Number of pages9
JournalJournal of Molecular Diagnostics
Volume3
Issue number4
DOIs
StatePublished - Nov 2001

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ASJC Scopus subject areas

  • Pathology and Forensic Medicine
  • Molecular Medicine

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