Increased DNA microarray hybridization specificity using sscDNA targets.
Increased DNA microarray hybridization specificity using sscDNA targets.
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DOI：
10.1186/1471-2164-6-57
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发表时间：
2005-04-22
期刊：
影响因子：
4.4
通讯作者：
Erle DJ
中科院分区：
文献类型：
--
作者：
Barker CS;Griffin C;Dolganov GM;Hanspers K;Yang JY;Erle DJ
基因表达微阵列分析中使用最广泛的扩增方法是使用T7 RNA聚合酶驱动的体外转录（IVT）来产生互补RNA (cRNA)，这些互补RNA可以杂交到阵列中。然而，当分析非常少量的起始RNA时，需要进行多轮扩增。此外，某些rna - dna错配比类似的dna - dna错配更稳定，这可能会增加非特异性杂交。我们试图确定最近开发的产生单链cDNA的线性等温扩增方法（核糖- spia）是否比传统的基于ivt的基于微阵列的转录物表达分析方法具有优势。当起始总RNA仅为5ng时，单轮核糖- spia扩增产生足够的sscDNA进行杂交。从重复扩增中获得的探针集信号强度的比较显示出一致的高相关性（r = 0.99）。我们使用核糖核酸- spia比较了两种不同人类RNA样本中的基因表达。与一轮IVT相比，尽管我们使用的起始RNA减少了1000倍，但核糖核酸- spia具有更大的动态范围，与定量PCR结果的相关性更好。改进的动态范围与对失配控制探针的杂交减少有关。使用扩增的sscDNA可能比基于ivt的扩增方法具有实质性的优势，特别是在起始RNA数量非常有限的情况下。使用sscDNA靶点而不是cRNA靶点似乎可以提高杂交特异性。
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. 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. 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.
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