ABSTRACT
The life of an mRNA is dynamic within a cell. The development of quantitative fluorescent microscopy techniques to image single molecules of RNA has allowed many aspects of the mRNA lifecycle to be directly observed in living cells. Recent advances in live-cell multicolor RNA imaging, however, have now made it possible to investigate RNA metabolism in greater detail. In this chapter, we present an overview of the design and implementation of the translating RNA imaging by coat protein knockoff RNA biosensor, which allows untranslated mRNAs to be distinguished from ones that have undergone a round of translation. The methods required for establishing this system in mammalian cell lines and Drosophila melanogaster oocytes are described here, but the principles may be applied to any experimental system.
Subject(s)
Biosensing Techniques/methods , Drosophila melanogaster/cytology , Microscopy, Fluorescence/methods , Oocytes/cytology , RNA, Messenger/analysis , Animals , Capsid Proteins/genetics , Cells, Cultured , Drosophila melanogaster/genetics , Levivirus/genetics , Luminescent Proteins/genetics , Molecular Imaging/methods , Oocytes/metabolism , RNA, Messenger/geneticsABSTRACT
The quantitative release of creatine kinase (CK-MB) into the circulation of 97 patients receiving between three and five distal aorto-coronary bypass grafts was used to quantitate the minimal operative myocardial injury and to determine the diagnostic utility of this measurement in the detection of perioperative myocardial infarction. Independently read electrocardiography (ECG) was used to define infarction. The +/- SD confidence range for traumatized heart tissue based on 88 patients without infarction was 0-40.5 g equivalents. Six patients with perioperative myocardial infarction had values significantly above this range. Three patients with indeterminate ECG both released CK-MB significantly above the reference range and were clearly abnormal from a clinical standpoint.
