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BMC Genomics

, 6:57

First Online: 22 April 2005Received: 17 February 2005Accepted: 22 April 2005

Abstract

BackgroundThe 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.

ResultsA 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.

ConclusionThe 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.

Electronic supplementary materialThe online version of this article doi:10.1186-1471-2164-6-57 contains supplementary material, which is available to authorized users.

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Author: Christopher S Barker - Chandi Griffin - Gregory M Dolganov - Kristina Hanspers - Jean Yee Hwa Yang - David J Erle

Source: https://link.springer.com/article/10.1186/1471-2164-6-57







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