Purification of transcripts and metabolites from Drosophila heads.
Purification of transcripts and metabolites from Drosophila heads.
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DOI：
10.3791/50245
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发表时间：
2013-03-15
期刊：
影响因子：
--
通讯作者：
Fernandez-Funez P
中科院分区：
文献类型：
--
作者：
Jensen K;Sanchez-Garcia J;Williams C;Khare S;Mathur K;Graze RM;Hahn DA;McIntyre LM;Rincon-Limas DE;Fernandez-Funez P
在过去的十年里，我们试图了解神经元变性的分子和细胞机制，使用果蝇作为模式生物。虽然果蝇提供了明显的实验优势，但对神经退行性疾病的研究主要依赖于传统技术，包括遗传相互作用，组织学，免疫荧光和蛋白质生物化学。这些技术对于机械的、假设驱动的研究是有效的，这些研究导致对单个基因在明确定义的生物学问题中的作用的详细理解。然而，神经退行性疾病是高度复杂的，并随着时间的推移影响多个细胞器和过程。新技术和组学时代的到来为了解复杂疾病背后的全球细胞扰动提供了一个独特的机会。灵活的模式生物，如果蝇，是适应这些新技术的理想选择，因为它们具有很强的注释性和高度的可处理性。然而，这些小动物面临的一个挑战是从高度相关的组织（如苍蝇大脑）中纯化足够的信息分子（DNA，mRNA，蛋白质，代谢物）。其他挑战包括收集大量的苍蝇进行实验复制（对统计稳健性至关重要），并制定一致的程序来纯化高质量的生物材料。在这里，我们描述了收集数千个苍蝇头和提取转录物和代谢物的程序，以了解基因表达和代谢的全球变化如何导致神经退行性疾病。这些程序易于扩展，可应用于研究蛋白质组学和表观基因组学对疾病的贡献。
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.
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