Ghorbel Mohamed T, Murphy David
Bristol Heart Institute, University of Bristol, Bristol, UK.
Methods Mol Biol. 2011;789:237-59. doi: 10.1007/978-1-61779-310-3_15.
Comparing two RNA populations that differ from the effects of a single independent variable, such as a drug treatment or a specific genetic defect, can establish differences in the abundance of specific transcripts that vary in a population dependent manner. There are different methods for identifying differentially expressed genes. These methods include microarray, Serial Analysis of Gene Expression (SAGE), and quantitative Reverse-Transcriptase Polymerase Chain Reaction (qRT-PCR). Herein, the protocol describes an easy and cost-effective alternative that does not require prior knowledge of the transcriptomes under examination. It is specifically relevant when low levels of RNA starting material are available. This protocol describes the use of Switching Mechanism At RNA Termini Polymerase Chain Reaction (SMART-PCR) to amplify cDNA from small amounts of RNA. The amplified cDNA populations under comparison are then subjected to Suppression Subtractive Hybridization (SSH-PCR). SSH-PCR is a technique that couples subtractive hybridization with suppression PCR to selectively amplify fragments of differentially expressed genes. The resulting products are cDNA populations enriched for significantly overrepresented transcripts in either of the two input RNAs. These cDNA populations can then be cloned to generate subtracted cDNA library. Microarrays made with clones from the subtracted forward and reverse cDNA libraries are then screened for differentially expressed genes using targets generated from tester and driver total RNAs.
比较两个因单一自变量（如药物治疗或特定基因缺陷）的影响而不同的RNA群体，可以确定特定转录本丰度的差异，这些差异以群体依赖的方式变化。有多种鉴定差异表达基因的方法。这些方法包括微阵列、基因表达序列分析（SAGE）和定量逆转录聚合酶链反应（qRT-PCR）。在此，本方案描述了一种简单且经济高效的替代方法，该方法不需要对所检测的转录组有先验知识。当起始RNA材料水平较低时，该方法尤为适用。本方案描述了使用RNA末端转换机制聚合酶链反应（SMART-PCR）从少量RNA中扩增cDNA。然后，将扩增后的用于比较的cDNA群体进行抑制性消减杂交（SSH-PCR）。SSH-PCR是一种将消减杂交与抑制性PCR相结合的技术，用于选择性扩增差异表达基因的片段。所得产物是富含两个输入RNA中任一显著高表达转录本的cDNA群体。然后可以将这些cDNA群体进行克隆以生成消减cDNA文库。然后使用由测试者和驱动者总RNA产生的靶标，筛选由消减正向和反向cDNA文库的克隆制成微阵列，以鉴定差异表达基因。
