Whole Transcriptome Amplification for Gene Expression Profiling and Development of Molecular Archives
| Autor: | Scott A. Tomlins, Rohit Mehra, Daniel R. Rhodes, Rajal B. Shah, Mark A. Rubin, Eric Bruening, Vladimir Makarov, Arul M. Chinnaiyan |
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| Jazyk: | angličtina |
| Rok vydání: | 2006 |
| Předmět: | |
| Zdroj: | Neoplasia: An International Journal for Oncology Research, Vol 8, Iss 2, Pp 153-162 (2006) |
| Druh dokumentu: | article |
| ISSN: | 1476-5586 1522-8002 |
| DOI: | 10.1593/neo.05754 |
| Popis: | 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. |
| Databáze: | Directory of Open Access Journals |
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