Methods in molecular exercise physiology

In this chapter, we describe and explain some of the most important methodology that molecular exercise physiologists use, at level of the molecule and cell, to investigate the microscopic make-up of our cell’s response to exercise. We discuss the important role of blood and muscle biopsy sampling and describe and explain, in detail, the different types of analysis that might be undertaken by molecular exercise physiologists at the molecular (DNA, RNA, protein), cellular and tissue levels. From a muscle tissue biopsy, we describe the process of isolating DNA, RNA, protein and processing of tissue/cells for immunolabelling analysis, as well as primary muscle-derived cell isolation for growing muscle cells in a dish (in-vitro culture). We describe and explain the methods used to analyse a person’s inherited genetic code and sequence variation of DNA between individuals at both the single/targeted gene level and across the genome (genomics), and how to investigate epigenetic modifications to DNA and chromatin again, at both the individual gene level and across the genome (epigenomics). We describe and explain the methods for determining whether our genes are turned on or off in response to exercise at the single gene level and across the ‘transcriptome’ (transcriptomics). We describe and explain the methods for analyses of protein, to measure the abundance and activity of proteins and their role in ‘signal transduction’ in response to exercise at the individual protein level and across the ‘proteome’ (proteomics). We also include a description of the most important immunolabelling methods assessing changes at the protein level in skeletal muscle cell/tissue level that may help confirm the changes (or cellular location of those changes) observed at the molecular level. Finally, we will provide a narrative for the technique of primary muscle cell isolation from human biopsies, and how these isolated cells can be used as ‘in a dish’ (in-vitro) culture models to mimic exercise (in both monolayer and three-dimensional cultures) to compliment molecular analyses from whole-body (in-vivo) human exercise research.