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JMD 2004, Vol. 6, No. 1
Copyright © 2004 American Society for Investigative Pathology & Association for Molecular Pathology

Utility of Linearly Amplified RNA for RT-PCR Detection of Chromosomal Translocations

Validation Using the t(2;5)(p23;q35) NPM-ALK Chromosomal Translocation

Jonathan A. Schumacher^*, Stephen D. Jenson, Kojo S. J. Elenitoba-Johnson^* and Megan S. Lim^*

From the Associated Regional and University Pathologists (ARUP) Institute for Clinical and Experimental Pathology * and the Department of Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah

The requirement for sufficient quantities of starting RNA has limited the ability to evaluate multiple transcripts using reverse transcriptase-polymerase chain reaction (RT-PCR). In this study, we demonstrate the utility of linear RNA amplification for RT-PCR analysis of multiple gene transcripts including a chromosomal translocation, using the t(2;5)(p23;q35) as a model. RNA from the t(2;5)-positive cell line, SU-DHL-1, and the t(2;5)-negative cell line, HUT-78, was extracted and exposed to two rounds of linear amplification. RT-PCR using cDNA from the resultant amplified (a) RNA and total RNA resulted in the 177 bp NPM-ALK fusion gene product from the SU-DHL-1 cell line, but not from aRNA or total RNA from the HUT-78 cell line. DNA sequencing of the RT-PCR products from total and aRNA of SU-DHL-1 cells demonstrated identical sequences corresponding to the NPM-ALK fusion gene. Evaluation of 25 snap-frozen tissue samples, including eight NPM-ALK-positive ALCLs demonstrated 100% concordance of t(2;5) detection between cDNA from total RNA and that from aRNA. Our results show that linear amplification of RNA can enhance starting RNA greater than 200-fold and can be used for rapid and specific detection of multiplex gene expression from a variety of sources. This method can generate a renewable archive of representative cDNA, which can be used for retrospective screening of stored samples as well as positive controls for the clinical molecular diagnostic laboratory.

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