Chapter 18 Methodological approaches to metabolism research

Abstract

This chapter discusses the principles, approaches, and applications of the molecular and metabolic techniques. Advances in molecular biology and stable isotope techniques provide an understanding of the biological basis of metabolomics from the level of systemic physiology, to intermediary metabolism, to molecular regulation of critical proteins, and on down to genomic expression. The development of transgenic technologies, such as gene overexpression, knockout, and conditional expression, the physiological roles and metabolic functions of specific genes are explored and individual proteins involved in the control of specific aspects of metabolism are identified. Metabolomics is feasible in crude biological extracts with advances in nuclear magnetic resonance spectroscopy, mass spectrometry coupled with bioinformatics techniques, and multivariate statistical analyses. Combination of laser capture microdissection with real-time quantitative real-time polymerase chain reaction (RT-PCR) allows expression of selected genes to be quantified in a pure population of defined individual cells. RNA interference is used as a specific and efficient method to silence gene expression in mammalian cells and to confirm gene function on a wholegenome scale.