Isolation and Amplification of Fungal RNA for Microarray Analysis from Host Samples
Transcriptional profiling is a powerful tool to investigate the interplay between pathogens and their hosts. For several pathogenic fungi, like Candida albicans, genome-wide microarrays are now available, and alternative methods, such as Serial Analysis o
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1. Introduction Isolation of RNA is essential for measuring gene expression at the transcriptional level. High throughput RNA-based technologies, such as microarrays, can provide important information about transcriptional changes, for example, during host–pathogen interactions (1). For such transcriptional profiling technologies, it is essential to isolate a sufficient amount of high-quality RNA, without DNA and protein contamination. For microarray analysis, at least 1 μg of RNA is generally considered necessary. However, if the starting material is limited, it is possible to overcome the requirement for large quantities of RNA by sophisticated amplification techniques. There are several protocols for amplifying RNA, mostly relying on T7-based anti-sense RNA amplification (2). RNA is a relatively stable molecule, which can, nonetheless, be rapidly degraded by widespread and extremely stable RNase enzymes. This results in shorter RNA fragments, which can negatively impact upon microarray analyses and other downstream Alexandra C. Brand and Donna M. MacCallum (eds.), Host-Fungus Interactions: Methods and Protocols, Methods in Molecular Biology, vol. 845, DOI 10.1007/978-1-61779-539-8_28, © Springer Science+Business Media, LLC 2012
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applications. Furthermore, rare transcripts may be lost and may therefore not be detected in subsequent steps. One simple approach to determine RNA quality is to use lab-on-a-chip solutions such as the “Agilent 2100 Bioanalyzer.” These instruments allow quantification and quality control of RNA samples in a short time (3). Compared to most animal or bacterial cells, RNA isolation from fungi is more demanding. This is mainly due to the thick fungal cell wall, which is difficult to disrupt. However, because transcriptional changes can occur within minutes, disruption must be achieved in as little time as possible (4, 5). Therefore, speed is essential to ensure that RNA levels reflect the experimental condition of interest rather than a response to the isolation procedure itself (physical treatments such as centrifugation, chemicals, temperature, etc.). One commonly used method to rapidly disrupt fungal cell walls is treatment with phenol and SDS combined with freeze–thaw cycles. Another common and efficient technique is a high-speed homogenisation in the presence of glass beads and denaturing reagents. With samples from infection models, both methods will result in a mixture of host and pathogen RNA. However, some models allow removal of the bulk of host RNA by taking advantage of the different RNA isolation kinetics from host and fungal cells (described in Subheading 2.1). Nonetheless, in our experience host:pathogen RNA ratios of up to 5:1 are acceptable even for sensitive microarray applications ((6) and unpublished data). In this chapter, we describe two methods for total RNA extraction from host samples infected with pathogenic yeasts. The first protocol describes fungal RNA isolation from infected monolayer cells and the second deals with RNA isolation fr
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