By Erika J. Ernst
A suite of state of the art molecular equipment for learning antifungal resistance, for locating and comparing either new and latest antifungal medicinal drugs, and for knowing the host reaction and immunotherapy of such brokers. The protocols stick with the profitable tools in Molecular medication™ sequence layout, every one providing step by step laboratory directions, an advent outlining the main in the back of the approach, lists of the mandatory gear and reagents, and tips about troubleshooting and heading off recognized pitfalls. Antifungal brokers: equipment and Protocols deals clinician-scientists, microbiologists and molecular biologists the effective instruments they want this present day to appreciate and effectively improve new healing brokers for yeast, mildew, and fungal infections.
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Additional info for Antifungal Agents
54, 463–498. 2. , and Bille, J. (1996) Susceptibilities of Candida albicans multidrug transporter mutants to various antifungal agents and other metabolic inhibitors. Antimicrob. Agents Chemother. 40, 2300–2305. 3. , and Staib, P. (1999) Sequential gene disruption in Candida albicans by FLP-mediated site-specific recombination. Mol Microbiol 32, 547–556. 4. , and Morschhäuser, J. (2000) Targeted gene disruption in Candida albicans wild-type strains: the role of the MDR1 gene in fluconazole resistance of clinical Candida albicans isolates.
QIA-quick nucleotide removal kit (containing Buffer PB, Buffer PE, and purification columns). Microcon-30 filter. Cy3-labeled deoxycytidine 5'-triphosphate (dCTP). Cy5-labeled dCTP. C. albicans microarray chips. Salmon sperm DNA. Hybridization buffer. Hybridization chamber. 3 M sodium citrate. Microarray scanner. 3. cDNA Synthesis and RT-PCR 1. 2. 3. 4. 5. 6. 7. 8. Oligo(dT) primer or random hexamer primers. RNase block ribonuclease inhibitor. Moloney murine leukemia virus reverse transcriptase.
The ratios between the two signals for each gene give an indication of relative gene expression. DNA microarray analysis is well suited for the assessment of differential gene expression between isolates within matched sets of azole-susceptible and -resistant C. albicans. Although we routinely perform experiments in replicate at minimum, we also confirm the differential expression of select genes independently using RT-PCR. Other methods, such as Northern blot or real-time RT-PCR, are also appropriate.