Single-stranded linear amplification protocol results in reproducible and reliable microarray data from nanogram amounts of starting RNA.
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.
Stirewalt
Derek L
DL
Clinical Research Division, Fred Hutchinson Cancer Research Center, University of Washington, Seattle, WA 98109, USA. dstirewa@fhcrc.org
Pogosova-Agadjanyan
Era L
EL
Khalid
Najma
N
Hare
Dan R
DR
Ladne
Paula A
PA
Sala-Torra
Olga
O
Zhao
Lue Ping
LP
Radich
Jerald P
JP
D001710:Biotin; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods* / Q000592:standards; D020411:Oligonucleotide Array Sequence Analysis; D015203:Reproducibility of Results
D001710:Biotin