Transcript Profiling Using ESTs from Paracoccidioides brasiliensis in Models of Infection
Transcript profiling is an invaluable strategy to study differential gene expression. Here we describe a detailed protocol for applying a subtractive hybridization technique, representational difference analysis (RDA), as a molecular strategy for the iden
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1. Introduction Representational difference analysis (RDA) couples subtractive hybridization to PCR-mediated kinetic enrichment for the detection of differences between genomes and transcriptomes. This method, first described by Lisitsyn and coworkers (1), was initially applied to the detection of differences between two genomes. Subsequently, Hubank and Schatz (2) adapted RDA for use with complementary DNA (cDNA), with Pastorian and coworkers (3) optimizing the methodology for identification of differentially expressed mRNAs. Differentially expressed cDNA sequences in two distinct populations (one designated driver and the other tester) are determined based upon a combination of subtractive hybridization and PCR amplification of the fragments present in one population but not in other. Following these steps, those
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_26, © Springer Science+Business Media, LLC 2012
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fragments present in both populations are eliminated, leaving only the differentially expressed populations. This technique is a good strategy to be used when microarrays and/or genome sequences are not available. Besides that, RDA is cheaper and fast expression analysis method. In this chapter, we describe an application of the RDA methodology in the search for genes potentially relevant to pathogenesis of the pathogenic dimorphic fungus, Paracoccidioides brasiliensis. The methodology described has been adapted from previous RDA studies (3) and provides a through description of the RDA method employed to identify differentially expressed genes in yeast cells derived from infected mice and from ex vivo models of infection. The detailed laboratory protocol provides a step-by-step description of the procedure and includes a description of tools that can be used in the bioinformatic analysis of the obtained expressed sequence tags (ESTs).
2. Materials All solutions and reagents should be prepared and stored at room temperature, unless indicated otherwise. Dispose all used materials following waste disposal regulations. 2.1. Reagents for Infection Models
1. P. brasiliensis strain/isolate. 2. 40% Glucose solution. Sterilize by filtration. 3. Gentamicin (10 mg/mL). 4. Fava-Netto’s medium. Add 400 mL water to a 1-L beaker. Add 10 g bacteriological peptone, 5 g yeast extract, 3 g proteose peptone, 5 g beef extract, and 5 g NaCl. Adjust the volume to 900 mL with water. Mix and adjust pH to 7.2. Sterilize by autoclaving. Cool and add 1 mL gentamicin and 100 mL glucose solution. For semisolid medium, add 10 g agar prior to autoclaving. 5. 4–6-week-old BALB/c mice. 6. Isotonic saline (0.9% NaCl) solution. 7. Syringes (3 mL) and needles (22G1"). 8. Tissue grinder or homogenizer. 9. Organ culture medium. Add 400 mL water to a 1-L graduated container. Add 50 g of Brain and Heart Infusion (BHI) broth medium. Add water to 800 mL, mix, and adjust pH to 7.0. Add 10 g agar and m
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