Whole transcriptome amplification for gene expression profiling and development of molecular archives

被引:39
作者
Tomlins, Scott A.
Mehra, Rohit
Rhodes, Daniel R.
Shah, Rajal B.
Rubin, Mark A.
Bruening, Eric
Makarov, Vladimir
Chinnaiyan, Arul M.
机构
[1] Univ Michigan, Sch Med, Dept Pathol, Ann Arbor, MI 48109 USA
[2] Univ Michigan, Sch Med, Dept Bioinformat, Ann Arbor, MI 48109 USA
[3] Univ Michigan, Sch Med, Dept Urol, Ann Arbor, MI 48109 USA
[4] Harvard Univ, Brigham & Womens Hosp, Sch Med, Dept Pathol, Boston, MA 02115 USA
[5] Rubicon Genom Inc, Ann Arbor, MI 48108 USA
[6] Univ Michigan, Sch Med, Michigan Urol Ctr, Ann Arbor, MI 48109 USA
[7] Univ Michigan, Sch Med, Ctr Comprehens Canc, Ann Arbor, MI 48109 USA
来源
NEOPLASIA | 2006年 / 8卷 / 02期
关键词
exponential RNA amplification; whole transcriptome amplification; prostate cancer; laser capture microdissection; FFPE tissue;
D O I
10.1593/neo.05754
中图分类号
R73 [肿瘤学];
学科分类号
100214 ;
摘要
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.
引用
收藏
页码:153 / 162
页数:10
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