Suppression Subtractive Hybridization
Suppression Subtractive Hybridization
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DOI：
10.1007/978-1-61779-310-3_15
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发表时间：
2011-01-01
期刊：
影响因子：
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通讯作者：
Murphy, David
中科院分区：
文献类型：
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作者：
Ghorbel, Mohamed T.;Murphy, David
比较两个不同于单一自变量影响的RNA群体，如药物治疗或特定的遗传缺陷，可以建立特定转录本丰度的差异，这些转录本的丰度以群体依赖的方式变化。有不同的方法来识别差异表达的基因。这些方法包括基因芯片、基因表达系列分析(SAGE)和定量逆转录聚合酶链式反应(qRT-PCR)。在此，该方案描述了一种简单且成本效益高的替代方案，该方案不需要事先了解正在检查的转录本。当有低水平的RNA起始物质可用时，这一点特别相关。本协议描述了利用RNA末端切换机制聚合酶链式反应(SMART-PCR)从少量RNA中扩增出cDNA.然后对比较中的扩增的cDNA群进行抑制消减杂交(SSH-PCR)。SSH-PCR是一种将消减杂交与抑制性聚合酶链式反应相结合的技术，用于选择性地扩增差异表达基因片段。由此产生的产物是在两个输入RNA中的任何一个中显著过度表达转录本的cDNA群。然后克隆这些cDNA群，以产生消减的cDNA库。然后，使用从测试者和驱动器总RNA产生的靶标，从消减的正向和反向cDNA文库中克隆制成的微阵列，筛选差异表达基因。
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.