Quantitative Assessment of Fungal Burden in Murine Brains

Quantitative PCR was used to assess fungal burden in brains after systemic infection with C. albicans as previously described^{32 (link)}. Brains were harvested from mice transcardially perfused with 20 mL of ice-cold PBS and placed directly into lysis buffer from DNeasy Blood and Tissue Kit (Qiagen) and homogenized with tissue homogenizer (MP Biomedicals). Total DNA from 20 μg of brain homogenate was extracted using DNeasy Blood and Tissue Kit (Qiagen). Using a CFX96 Real-Time PCR machine (BioRad Laboratories), real-time PCR was performed with 40 ng of DNA per sample using SYBR Green (Applied Biosystems) or water (non-template negative control) with the following C. albicans-specific primers: forward primer - ACT TCT GTA AGA GTG CTG GTTC and reverse primer - GCA TGC CAG GAG AGT GTA AA (Integrated DNA Technologies). The following cycling conditions were used: an initial denaturation step of 95°C for 10 minutes followed by 40 cycles of 95°C for 5 seconds and 60°C for 30 seconds. Reactions were performed in duplicates and with generation of melting curves to confirm purity post DNA amplification. The number of C. albicans genome copies was quantified by normalizing against a series of standards that consisted of DNA isolated from brain homogenates from uninfected mice spiked with known CFU quantities of C. albicans.