Whole transcriptome amplification for gene expression profiling and development of molecular archives.
Neoplasia, 2006/2;8(2):153-62.
Tomlins SA[1], Mehra R, Rhodes DR, Shah RB, Rubin MA, Bruening E, Makarov V, Chinnaiyan AM
Affiliations
PMID: 16611408
Impact factor: 6.218
Abstract
Expression profiling of clinically obtainable tumor specimens has been hindered by the need for microgram quantities of RNA. In vitro transcription (IVT)-based amplifications are most commonly used to amplify small quantities of RNA for microarray analysis. However, significant drawbacks exist with IVT-based amplification, and the need for alternative amplification methods remains. Herein, we validate whole transcriptome amplification (WTA), an exponential amplification technique that produces cDNA libraries and amplified target in 3 to 4 hours from nanogram quantities of total RNA using a combination of cDNA microarrays and quantitative polymerase chain reaction (PCR). We demonstrate that WTA material can serve as a "molecular archive" because a WTA cDNA library can be faithfully amplified through multiple rounds of PCR amplification, allowing it to serve as a bankable and distributable resource. To demonstrate applicability, WTA was combined with laser capture microdissection to profile frozen prostate tissues. Unlike most IVT-based and exponential amplification techniques, WTA does not depend on the presence of a poly-A tail. Thus, we demonstrate that WTA is compatible with artificially degraded RNA and RNA isolated from formalin-fixed paraffin-embedded tissues. Taken together, WTA represents a versatile approach to profile and archive cDNA from minute tumor samples and is compatible with partially degraded RNA.
MeSH terms
Biological Specimen Banks; Gene Amplification; Gene Expression Profiling; Genome, Human; Humans; Models, Genetic; Neoplasms; Polymerase Chain Reaction; RNA, Neoplasm; Transcription, Genetic
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