title: Single-stranded linear amplification protocol results in reproducible and reliable microarray data from nanogram amounts of starting RNA.
abstract: The range of scientific questions utilizing DNA microarray techniques is limited by the fact that these methods require 5-40 microg 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 microg 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 microg. These results suggest that SLAP is an excellent alternative to IVT-based amplification protocols when RNA is limited by small sample size.
authors.affiliation: Clinical Research Division, Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA 98109, USA. dstirewa@fhcrc.org
authors.forename: Derek L
authors.initials: DL
authors.lastname: Stirewalt
authors.forename: Era L
authors.initials: EL
authors.lastname: Pogosova-Agadjanyan
authors.forename: Najma
authors.initials: N
authors.lastname: Khalid
authors.forename: Dan R
authors.initials: DR
authors.lastname: Hare
authors.forename: Paula A
authors.initials: PA
authors.lastname: Ladne
authors.forename: Olga
authors.initials: O
authors.lastname: Sala-Torra
authors.forename: Lue Ping
authors.initials: LP
authors.lastname: Zhao
authors.forename: Jerald P
authors.initials: JP
authors.lastname: Radich
mesh_terms: D001710:Biotin; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods* / Q000592:standards; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results
chemical_list: D001710:Biotin