title: Whole transcriptome amplification for gene expression profiling and development of molecular archives.
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.
authors.affiliation: Department of Pathology, University of Michigan Medical School, Ann Arbor, MI 48109-0602, USA.
authors.forename: Scott A
authors.initials: SA
authors.lastname: Tomlins
authors.forename: Rohit
authors.initials: R
authors.lastname: Mehra
authors.forename: Daniel R
authors.initials: DR
authors.lastname: Rhodes
authors.forename: Rajal B
authors.initials: RB
authors.lastname: Shah
authors.forename: Mark A
authors.initials: MA
authors.lastname: Rubin
authors.forename: Eric
authors.initials: E
authors.lastname: Bruening
authors.forename: Vladimir
authors.initials: V
authors.lastname: Makarov
authors.forename: Arul M
authors.initials: AM
authors.lastname: Chinnaiyan
mesh_terms: D018070:Biological Specimen Banks; D005784:Gene Amplification; D020869:Gene Expression Profiling; D015894:Genome, Human; D006801:Humans; D008957:Models, Genetic; D009369:Neoplasms / Q000235:genetics*; D016133:Polymerase Chain Reaction; D012334:RNA, Neoplasm / Q000235:genetics; D014158:Transcription, Genetic
chemical_list: D012334:RNA, Neoplasm