Journal
GENOMICS
Volume 83, Issue 2, Pages 321-331Publisher
ACADEMIC PRESS INC ELSEVIER SCIENCE
DOI: 10.1016/j.ygeno.2003.08.008
Keywords
microarray; in vitro transcription; affymetrix; RNA amplification; eberwine; array
Funding
- NCI NIH HHS [CA85053, K23 CA92405-01] Funding Source: Medline
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The range of scientific questions utilizing DNA microarray techniques is limited by the fact that these methods require 5-40 mug of high-quality total RNA. Thus, methods that reliably amplify the starting RNA amount could expand the applicability of DNA microarray technology. We developed a single-stranded linear amplification protocol (SLAP) that combines the reproducibility of in vitro transcription and the amplification robustness of polymerase chain reactions. We compared SLAP to the NIH-IVT amplification protocol. SLAP displayed excellent conservation of the 5'/3' signal and demonstrated the most robust amplification, producing the recommended amounts of biotin-labeled RNA with as little as 0.002 mug of starting RNA. Both SLAP and NIH-IVT methods demonstrated good reproducibility, but SLAP maintained the highest level of reliability with RNA starting amounts of <0.05 mug. These results suggest that SLAP is an excellent alternative to IVT-based amplification protocols when RNA is limited by small sample size. (C) 2003 Elsevier Inc. All rights reserved.
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