title: Utility of linearly amplified RNA for RT-PCR detection of chromosomal translocations: validation using the t(2;5)(p23;q35) NPM-ALK chromosomal translocation.
abstract: 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.
authors.affiliation: Associated Regional and University Pathologists Institute for Clinical and Experimental Pathology, University of Utah Health Sciences Center, Salt Lake City, Utah 84132, USA.
authors.forename: Jonathan A
authors.initials: JA
authors.lastname: Schumacher
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Jenson
authors.forename: Kojo S J
authors.initials: KS
authors.lastname: Elenitoba-Johnson
authors.forename: Megan S
authors.initials: MS
authors.lastname: Lim
mesh_terms: D045744:Cell Line, Tumor; D002889:Chromosomes, Human, Pair 2 / Q000235:genetics; D002895:Chromosomes, Human, Pair 5 / Q000235:genetics; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D015514:Oncogene Proteins, Fusion / Q000235:genetics; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D014178:Translocation, Genetic
chemical_list: D015514:Oncogene Proteins, Fusion; C107023:p80(NPM-ALK) protein; D011505:Protein-Tyrosine Kinases