Global amplification of mRNA by template-switching PCR: linearity and application to microarray analysis
Global amplification of mRNA by template-switching PCR: linearity and application to microarray analysis
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DOI：
10.1093/nar/gng142
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发表时间：
2003-11-15
影响因子：
14.9
通讯作者：
Lyons, PA
中科院分区：
文献类型：
--
作者：
Petalidis, L;Bhattacharyya, S;Lyons, PA
用于表达微阵列分析的传统目标标记方法通常需要相对大量的总 RNA，当可用样本较少时，这是一个严重的限制。在这里，我们探讨了模板转换 PCR 的循环依赖性扩增特性，并验证了其在微阵列靶标标记中的用途。 TS-PCR 可鉴定高达 80% 的差异表达基因，这些差异表达基因是通过直接标记使用更少 30 倍的输入 RNA 进行扩增而鉴定的，相当于每次杂交使用的起始材料少了 1000 倍。此外，微阵列实验的灵敏度显着提高，允许使用直接标记制备的靶标来鉴定低于检测水平的差异表达转录本。我们还验证了扩增的保真度，并表明扩增材料忠实地代表了起始 mRNA 群体。该方法不仅在灵敏度和差异表达基因的识别方面优于传统的标记策略，而且比其他扩增方案更快、劳动强度更低。
Conventional approaches to target labelling for expression microarray analysis typically require relatively large amounts of total RNA, a serious limitation when the sample available is small. Here we explore the cycle-dependent amplification characteristics of Template-Switching PCR and validate its use for microarray target labelling. TS-PCR identifies up to 80% of the differentially expressed genes identified by direct labelling using 30-fold less input RNA for the amplification, with the equivalent of 1000-fold less starting material being used for each hybridisation. Moreover, the sensitivity of microarray experiments is increased considerably, allowing the identification of differentially expressed transcripts below the level of detection using targets prepared by direct labelling. We have also validated the fidelity of amplification and show that the amplified material faithfully represents the starting mRNA population. This method outperforms conventional labelling strategies, not only in terms of sensitivity and the identification of differentially expressed genes, but it is also faster and less labour intensive than other amplification protocols.