| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
mRNA Using Real-Time Reverse-Transcription Polymerase Chain Reaction
From the Department of Medicine,
*
Roswell Park Cancer Institute, Buffalo, New York; Applied Biosystems,
Foster City, California; the Department of Pathology,
University of New Mexico Cancer Research Facility, Albuquerque, New Mexico; and the Department of Oncology,
Montefiore Medical Center and Albert Einstein Cancer Center, Bronx, New York
We have developed a sensitive and quantitative
reverse-transcription polymerase chain reaction (RT-PCR) assay for
detection of PML-RAR
, 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-RAR
plasmid DNA. PML-RAR 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-RAR-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.
This article has been cited by other articles:
 |
|
 |
|
  C. Santamaria, M. C. Chillon, C. Fernandez, P. Martin-Jimenez, A. Balanzategui, R. Garcia Sanz, J. F. San Miguel, and M.-G. Gonzalez Using quantification of the PML-RAR{alpha} transcript to stratify the risk of relapse in patients with acute promyelocytic leukemia Haematologica, March 1, 2007; 92(3): 315 - 322. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Schulz, T. Hyslop, J. Haaf, C. Bonaccorso, K. Nielsen, M. E. Witek, R. Birbe, J. Palazzo, D. Weinberg, and S. A. Waldman A Validated Quantitative Assay to Detect Occult Micrometastases by Reverse Transcriptase-Polymerase Chain Reaction of Guanylyl Cyclase C in Patients with Colorectal Cancer Clin. Cancer Res., August 1, 2006; 12(15): 4545 - 4552. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. E. Gallagher, B. Y. Yeap, W. Bi, K. J. Livak, N. Beaubier, S. Rao, C. D. Bloomfield, F. R. Appelbaum, M. S. Tallman, J. L. Slack, et al. Quantitative real-time RT-PCR analysis of PML-RARalpha mRNA in acute promyelocytic leukemia: assessment of prognostic significance in adult patients from intergroup protocol 0129 Blood, April 1, 2003; 101(7): 2521 - 2528. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Ovstebo, K. B. F. Haug, K. Lande, and P. Kierulf PCR-based Calibration Curves for Studies of Quantitative Gene Expression in Human Monocytes: Development and Evaluation Clin. Chem., March 1, 2003; 49(3): 425 - 432. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. M. Murphy, K. K. O. Watt, D. Cameron-Smith, C. J. Gibbons, and R. J. Snow Effects of creatine supplementation on housekeeping genes in human skeletal muscle using real-time RT-PCR Physiol Genomics, January 15, 2003; 12(2): 163 - 174. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
T. L. Applegate, H. J. Iland, E. Mokany, and A. V. Todd Molecular Monitoring of Acute Promyelocytic Leukemia by DzyNA Reverse Transcription-PCR Clin. Chem., October 1, 2002; 48(10): 1858 - 1860. [Full Text] [PDF]
|
|
|
|
|
|
T. L. Applegate, H. J. Iland, E. Mokany, and A. V. Todd Diagnosis and Molecular Monitoring of Acute Promyelocytic Leukemia Using DzyNA Reverse Transcription-PCR to Quantify PML/RAR{alpha} Fusion Transcripts Clin. Chem., August 1, 2002; 48(8): 1338 - 1343. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
