Loos Martin, Künzli Beat, Friess Helmut
Department of Surgery, Technische Universität München, Munich, Germany.
Methods Mol Biol. 2010;576:351-62. doi: 10.1007/978-1-59745-545-9_18.
Within the past decade, the field of gene expression analysis has constantly evolved, with numerous technologies being available for RNA quantification, including differential display, serial analysis of gene expression (SAGE), quantitative real-time (qRT) polymerase chain reaction (PCR), and microarrays. Although every technique has its specific application, the high levels of accuracy, reproducibility, sensitivity, and specificity have established qRT-PCR as a standard method for detection and quantification of gene expression. In this chapter, all steps of the qRT-PCR procedure, including purification of total RNA from animal tissues, reverse transcription to complementary DNA (cDNA), and quantification of relative gene expression are discussed. We chose qRT-PCR analysis of CD39 in pancreatic tissue as an example that is applicable to any gene of interest. CD39/ecto-nucleoside triphosphate diphosphohydrolase-type-1 (ENTPD1) is the dominant vascular ecto-nucleotidase that hydrolyzes extracellular nucleotides to integrate purinergic signaling responses. It has recently been associated with tumor growth and proliferation in melanoma cells and linked to pancreatic cancer progression.
在过去十年中，基因表达分析领域不断发展，有多种技术可用于RNA定量，包括差异显示、基因表达序列分析（SAGE）、定量实时（qRT）聚合酶链反应（PCR）和微阵列。尽管每种技术都有其特定应用，但qRT-PCR凭借其高度的准确性、可重复性、灵敏度和特异性，已成为检测和定量基因表达的标准方法。在本章中，将讨论qRT-PCR程序的所有步骤，包括从动物组织中纯化总RNA、逆转录为互补DNA（cDNA）以及相对基因表达的定量。我们选择胰腺组织中CD39的qRT-PCR分析作为一个适用于任何感兴趣基因的示例。CD39/胞外核苷三磷酸二磷酸水解酶-1型（ENTPD1）是主要的血管外切核苷酸酶，可水解细胞外核苷酸以整合嘌呤能信号反应。它最近与黑色素瘤细胞中的肿瘤生长和增殖相关，并与胰腺癌进展有关。
