Cell type-specific analysis of mRNA synthesis and decay in vivo with uracil phosphoribosyltransferase and 4-thiouracil.
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.
Cleary
Michael D
MD
University of California, Merced School of Natural Sciences, Merced, California, USA.
D000818:Animals; D015870:Gene Expression; D006801:Humans; D009928:Organ Specificity; D010430:Pentosyltransferases / Q000378:metabolism*; D020871:RNA Stability; D012333:RNA, Messenger / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D013889:Thiouracil / Q000031:analogs & derivatives* / Q000378:metabolism
D012333:RNA, Messenger; C114719:4-thiouracil; D013889:Thiouracil; D010430:Pentosyltransferases; C023269:uracil phosphoribosyltransferase