Candida albicans Culture, Cell Harvesting, and Total RNA Extraction
Candida albicans Culture, Cell Harvesting, and Total RNA Extraction
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DOI：
10.21769/bioprotoc.3803
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发表时间：
2020-11-05
期刊：
影响因子：
0.8
通讯作者：
Mitchell, Aaron P.
中科院分区：
文献类型：
--
作者：
Cravener, Max, V;Mitchell, Aaron P.
转录分析已经成为生物学研究的基石，并且随着在过去十年中更便宜和更有效的测序技术的出现，存在对高产和有效的RNA提取技术的需求。真菌如人类病原体白色念珠菌（Candida albicans）以由许多不同组分如几丁质组成的坚韧细胞壁的形式存在对RNA纯化的独特障碍，所述几丁质对许多常见的哺乳动物或细菌细胞裂解方法具有抗性。典型的体外C.如果处理许多样品时有多个产品损失或样品变化的机会，则白色念珠菌细胞收获方法可能是耗时且昂贵的。通过真空过滤而不是离心收集细胞减少了细胞冷冻前的时间，因此减少了RNA表达谱改变的可用时间。真空过滤是C.白色念珠菌有两个主要原因：由于暴露的表面积增加，细胞裂解在非沉淀细胞上更快，并且丝状细胞首先难以沉淀，这与酵母或细菌细胞不同。与酶处理相比，通过氧化锆/二氧化硅珠粒使用机械细胞裂解减少了处理时间以及总成本。总的来说，该方法是一种快速、高效、高产的从体外培养的C.白色念珠菌
Transcriptional analysis has become a cornerstone of biological research, and with the advent of cheaper and more efficient sequencing technology over the last decade, there exists a need for high-yield and efficient RNA extraction techniques. Fungi such as the human pathogen Candida albicans present a unique obstacle to RNA purification in the form of the tough cell wall made up of many different components such as chitin that are resistant to many common mammalian or bacterial cell lysis methods. Typical in vitro C. albicans cell harvesting methods can be time consuming and expensive if many samples are being processed with multiple opportunities for product loss or sample variation. Harvesting cells via vacuum filtration rather than centrifugation cuts down on time before the cells are frozen and therefore the available time for the RNA expression profile to change. Vacuum filtration is preferred for C. albicans for two main reasons: cell lysis is faster on non-pelleted cells due to increased exposed surface area, and filamentous cells are difficult to pellet in the first place unlike yeast or bacterial cells. Using mechanical cell lysis, by way of zirconia/silica beads, cuts down on time for processing as well as overall cost compared to enzymatic treatments. Overall, this method is a fast, efficient, and high-yield way to extract total RNA from in vitro cultures of C. albicans.