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

Citation
Jl. Slack et al., Pre-clinical validation of a novel, highly sensitive assay to detect PML-RAR alpha mRNA using real-time reverse-transcription polymerase chain reaction, J MOL DIAGN, 3(4), 2001, pp. 141-149
Citations number
28
Language
INGLESE
art.tipo
Article
Categorie Soggetti
Research/Laboratory Medicine & Medical Tecnology
Journal title
JOURNAL OF MOLECULAR DIAGNOSTICS
ISSN journal
1525-1578 → ACNP
Volume
3
Issue
4
Year of publication
2001
Pages
141 - 149
Database
ISI
SICI code
1525-1578(200111)3:4<141:PVOANH>2.0.ZU;2-J
Abstract
We have developed a sensitive and quantitative reverse-transcription polyme rase chain reaction (RT-PCR) assay for detection of PML-RA-R alpha, the fus ion oncogene present as a specific marker in > 99% of cases of acute promye locytic leukemia (APL). The assay is linear over at least 5 orders of magni tude of input DNA or RNA, and detects as few as 4 copies of PML-RAR alpha p lasmid DNA. PML-RA-R alpha transcripts could be detected in mixtures contai ning 2 to 5 pg of RNA from fusion-containing cells in a background of 1 mug of RNA from PML-RAR alpha -negative cells. Using 1.0 to 2.5 mug of input R NA, the sensitivity of the assay was between 10(-5) and 10(-6). Furthermore , determination of GAPDH copy number in each reaction allowed an accurate a ssessment of sample-to-sample variation In RNA quality and reaction efficie ncy, with consequent definition of a detection limit for each sample assaye d. Using an internal calibrator, assay precision was high, with coefficient s of variation between 10 and 20%. An interlaboratory study using coded sam ples demonstrated excellent reproducibility and high concordance between La boratories. This assay will be used to test the hypothesis that sensitive a nd 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.