title: Purification of transcripts and metabolites from Drosophila heads.
abstract: For the last decade, we have tried to understand the molecular and cellular mechanisms of neuronal degeneration using Drosophila as a model organism. Although fruit flies provide obvious experimental advantages, research on neurodegenerative diseases has mostly relied on traditional techniques, including genetic interaction, histology, immunofluorescence, and protein biochemistry. These techniques are effective for mechanistic, hypothesis-driven studies, which lead to a detailed understanding of the role of single genes in well-defined biological problems. However, neurodegenerative diseases are highly complex and affect multiple cellular organelles and processes over time. The advent of new technologies and the omics age provides a unique opportunity to understand the global cellular perturbations underlying complex diseases. Flexible model organisms such as Drosophila are ideal for adapting these new technologies because of their strong annotation and high tractability. One challenge with these small animals, though, is the purification of enough informational molecules (DNA, mRNA, protein, metabolites) from highly relevant tissues such as fly brains. Other challenges consist of collecting large numbers of flies for experimental replicates (critical for statistical robustness) and developing consistent procedures for the purification of high-quality biological material. Here, we describe the procedures for collecting thousands of fly heads and the extraction of transcripts and metabolites to understand how global changes in gene expression and metabolism contribute to neurodegenerative diseases. These procedures are easily scalable and can be applied to the study of proteomic and epigenomic contributions to disease.
authors.affiliation: Department of Neurology, McKnight Brain Institute, University of Florida, USA.
authors.forename: Kurt
authors.initials: K
authors.lastname: Jensen
authors.forename: Jonatan
authors.initials: J
authors.lastname: Sanchez-Garcia
authors.forename: Caroline
authors.initials: C
authors.lastname: Williams
authors.forename: Swati
authors.initials: S
authors.lastname: Khare
authors.forename: Krishanu
authors.initials: K
authors.lastname: Mathur
authors.forename: Rita M
authors.initials: RM
authors.lastname: Graze
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Hahn
authors.forename: Lauren M
authors.initials: LM
authors.lastname: McIntyre
authors.forename: Diego E
authors.initials: DE
authors.lastname: Rincon-Limas
authors.forename: Pedro
authors.initials: P
authors.lastname: Fernandez-Funez
mesh_terms: D000818:Animals; D004330:Drosophila / Q000033:anatomy & histology / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D005260:Female; D020869:Gene Expression Profiling / Q000379:methods*; D008297:Male; D023421:Models, Animal; D059467:Transcriptome