Oligonucleotide microarray analysis of aminoallyl-labeled cDNA targets from linear RNA amplification.
Single-stranded long oligonucleotide-based (50- to 70-mer) microarrays offer several advantages over conventional cDNA microarrays. These include the easy preparation of the probes, low cost of array production, and low cross-contamination during probe handling. However, the application of oligonucleotide microarrays for the analysis of global gene expression with small amounts of total RNA using the conventional oligo(dT)-T7 promoter-based amplification is hampered by the single-stranded nature (sense strand) of oligonucleotide probes in microarrays. In this report, we describe modified RNA amplification methods generating antisense-labeled cDNA targets and a successful application for oligonucleotide microarray gene expression analysis. In the first round, mRNA was amplified linearly with oligo(dT)24T7-primed reverse transcription and in vitro transcription by T7 RNA polymerase. In the second round, random 9-mer T3 primers and T3 RNA polymerase were used to generate sense-strand amplified RNA (aRNA). Fluorescently labeled cDNA targets were generated from the aRNA and hybridized to the oligonucleotide microarrays. Our data show that the amplification provides highly reproducible results, as evidenced by a significant correlation between the amplified and nonamplified samples. We also demonstrate that amplification of RNA derived from laser-microdissected tumor samples reproduced the gene expression profiles that were obtained from total RNA isolated from the same samples.
Kaposi-Novak
Pal
P
National Cancer Institute , National Institutes of Health, Bethesda, MD 20892-4262, USA.
Lee
Ju-Seog
JS
Mikaelyan
Arsen
A
Patel
Vyomesh
V
Thorgeirsson
Snorri S
SS
D000818:Animals; D019985:Benchmarking; D018076:DNA, Complementary; D005784:Gene Amplification; D020869:Gene Expression Profiling / Q000379:methods*; D066298:In Vitro Techniques; D051379:Mice; D008822:Mice, Transgenic; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D012313:RNA / Q000032:analysis*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D018076:DNA, Complementary; D012313:RNA