Cleary Michael D
University of California, Merced School of Natural Sciences, Merced, California, USA.
Methods Enzymol. 2008;448:379-406. doi: 10.1016/S0076-6879(08)02619-0.
Microarray-based analysis of mRNA expression has provided a genome-wide understanding of the genes and pathways involved in many biological processes. However, two limitations are often associated with traditional microarray experiments. First, standard methods of microarray analysis measure mRNA abundance, not mRNA synthesis or mRNA decay, and, therefore, do not provide any information regarding the mechanisms regulating transcript levels. Second, microarrays are often performed with mRNA from a mixed population of cells, and data for a specific cell-type of interest can be difficult to obtain. This chapter describes a method, referred to here as "4TU-tagging," which can be used to overcome these limitations. 4TU-Tagging uses cell type-specific expression of the uracil phosphoribosyltransferase gene of Toxoplasma gondii and the uracil analog 4-thiouracil (4TU) to selectively tag and purify RNA. Pulse-labeling of newly synthesized RNA with 4TU followed by a "chase" with unmodified uracil allows in vivo measurements of mRNA synthesis and decay in specific cells. Experimental design considerations for applying 4TU-tagging to different systems and protocols for cell type-specific RNA tagging, purification, and microarray analysis are covered in this chapter.
基于微阵列的mRNA表达分析为全面了解参与许多生物过程的基因和通路提供了可能。然而，传统的微阵列实验往往存在两个局限性。首先，微阵列分析的标准方法测量的是mRNA丰度，而非mRNA合成或mRNA降解，因此无法提供任何有关调节转录水平机制的信息。其次，微阵列实验通常使用来自混合细胞群体的mRNA，难以获得特定感兴趣细胞类型的数据。本章介绍了一种称为“4TU标记”的方法，可用于克服这些局限性。4TU标记利用弓形虫尿嘧啶磷酸核糖转移酶基因的细胞类型特异性表达和尿嘧啶类似物4-硫尿嘧啶（4TU）来选择性标记和纯化RNA。用4TU对新合成的RNA进行脉冲标记，随后用未修饰的尿嘧啶进行“追踪”，可以在体内测量特定细胞中mRNA的合成和降解。本章涵盖了将4TU标记应用于不同系统的实验设计考量，以及细胞类型特异性RNA标记、纯化和微阵列分析的方案。
