Increased DNA microarray hybridization specificity using sscDNA targets.
BACKGROUND
The most widely used amplification method for microarray analysis of gene expression uses T7 RNA polymerase-driven in vitro transcription (IVT) to produce complementary RNA (cRNA) that can be hybridized to arrays. However, multiple rounds of amplification are required when assaying very small amounts of starting RNA. Moreover, certain cRNA-DNA mismatches are more stable than the analogous cDNA-DNA mismatches and this might increase non-specific hybridization. We sought to determine whether a recently developed linear isothermal amplification method (ribo-SPIA) that produces single stranded cDNA would offer advantages over traditional IVT-based methods for microarray-based analyses of transcript expression.


RESULTS
A single round of ribo-SPIA amplification produced sufficient sscDNA for hybridizations when as little as 5 ng of starting total RNA was used. Comparisons of probe set signal intensities obtained from replicate amplifications showed consistently high correlations (r = 0.99). We compared gene expression in two different human RNA samples using ribo-SPIA. Compared with one round IVT, ribo-SPIA had a larger dynamic range and correlated better with quantitative PCR results even though we used 1000-fold less starting RNA. The improved dynamic range was associated with decreases in hybridization to mismatch control probes.


CONCLUSIONS
The use of amplified sscDNA may offer substantial advantages over IVT-based amplification methods, especially when very limited amounts of starting RNA are available. The use of sscDNA targets instead of cRNA targets appears to improve hybridization specificity.
Barker
Christopher S
CS
Gladstone Institute of Cardiovascular Disease, The J. David Gladstone Institutes, San Francisco, California 94158, USA. cbarker@gladstone.ucsf.edu
Griffin
Chandi
C
Dolganov
Gregory M
GM
Hanspers
Kristina
K
Yang
Jean Yee Hwa
JY
Erle
David J
DJ
D000818:Animals; D017931:DNA Primers / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004277:DNA, Single-Stranded / Q000235:genetics*; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D005821:Genetic Techniques; D023281:Genomics / Q000379:methods; D006801:Humans; D051379:Mice; D008810:Mice, Inbred C57BL; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000295:instrumentation / Q000379:methods*; D016133:Polymerase Chain Reaction; D012313:RNA / Q000737:chemistry / Q000378:metabolism; D018075:RNA, Complementary / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000378:metabolism
D017931:DNA Primers; D018076:DNA, Complementary; D004277:DNA, Single-Stranded; D018075:RNA, Complementary; D014764:Viral Proteins; D012313:RNA; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases