Evolution of normalizer genes for the estudy of Candida Albicans gene expression by qPCR

Abstract

The microorganisms are becoming increasingly resistant to antimicrobials and Candida albicans strains are resistant to antifungal thus becomes important and necessary to conduct studies to evaluate the effects of new therapeutic methods, such as photodynamic therapy (PDT) on virulence factors of C. albicans. To use real-time PCR in the evaluation of the expression of genes of C. albicans after application of PDT we need to establish what the best housekeeping gene for relative quantification. The objective of this study is to evaluate the housekeeping genes ACT1, LSC2, RIP1 and PMA1 and check which of these genes is the most stable for the assessment of gene expression of C. albicans after PDT. Clinical isolates of C. albicans that are used were isolated from patients with HIV (n=3) and a standard strain ATCC 18804, which are stored at the Laboratory of Microbiology and Immunology, of Institute of Science and Technology, UNESP - Univ Estadual Paulista, campus São José dos Campos. The absolute quantification of gene expression is related to the production of these genes in clinical samples and the reference strain test using real-time PCR. The presence and expression of genes are analyzed before and after PDT. We will use methylene blue (300 mM) with laser sensitized aluminum gallium arsenide - low power (visible red, 660 nm) and erythrosine (5 mM) with green LED (532 ± 10 nm) for PDT. Four experimental groups will be evaluated for each sample: a)F+L+: sensitization with the photosensitizer and light irradiation, b)F+L-: Only treatment with the photosensitizer, c)F-L+: Only light irradiation and d)F-L-: control group without sensitization with photosensitizer and absence of light. The results will be analyzed by the software of real-time equipment.