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Optimization of RNA-extraction for the study of human host : fungal pathogen interactions
Book - Dissertation
Vulvovaginal candidiasis (VVC) is a widespread vaginal infection most frequently caused by Candida albicans which normally inhabits genitourinary tracts of healthy individuals. VVC is considered to be the second most common vaginal infection after bacterial vaginosis and its incidence is increasing significantly due to widespread increased use of antibiotics. Importantly, VVC also occurs in healthy women and can be fatal in immunocompromised individuals. The infection may additionally become recurrent (RVVC) which is defined as more than three episodes of VVC in a year, severely reducing the quality of life. Unfortunately, the causal reasons for getting multiple recurrences are not fully understood. The number of transcriptomic studies to elucidate host pathogen interactions have increased in an attempt to unravel pathogenesis and immune control of the disease. However, although most studies have identified differently expressed genes from the host side, it is difficult to obtain sufficient RNA reads from the fungus in clinical samples, i.e., vaginal swabs, because fungal reads are underrepresented as compared to the human counterpart. In this context, RNA extraction improvements or selection of an RNA extraction method that maximizes the RNA amount obtained from fungal cells may be a first step on the path to the quantification of differently expressed genes of C. albicans. The storage medium in which clinical samples are preserved may also affect RNA extraction in terms of RNA quantity and quality, and this should maintain transcriptional profiles unmodified until RNA is extracted. In addition, cell lysis of the fungi is an essential process to guarantee high-efficient RNA extraction since the number of lysed cells impacts directly on the amount of RNA yielded. In this thesis, we compared different cell lysis methods and different methods for RNA extraction from C. albicans stored in RNAlater, a solution that protects RNA from its degradation and avoids gene expression changes at room temperature. After detailed comparison of different approaches, we established horizontal bead beating in RiboPure Lysis Buffer followed by RNA extraction with the RiboPure Yeast Kit as the most efficient procedure to obtain the highest RNA yield. In addition, although we showed that the use of RNAlater slightly reduced RNA yield, it still offered high quality and quantity RNA that may guarantee reliable host-pathogen interactions studies. We have also compared different procedures for RNA extraction in PBMCs and we obtained similar results. Storage of human cells in RNAlater resulted in reduced RNA yield but still can be suited for high quality and quantity RNA-demanding techniques such as RNA-sequencing (RNA-seq). In addition, the RiboPure Kit also offered the highest RNA yield which makes this kit appealing for dual RNA-seq focused on the pathogen and the host. Finally, we also demonstrated that high-quality fungal nucleic acids can be enriched through the lysis of RNAlater-stored human cells by means of RLT buffer, a commercial lysis buffer used in RNeasy kits, and subsequent centrifugation to discard the nucleic acids from the human lysed cells in the supernatant. Furthermore, the enrichment of fungal nucleic acids was verified in different mixes composed of C. albicans cells and PBMCs in ratios 1:1, 1:10 and 1:100 in order to simulate realistic fungus:human proportions encountered within the host.In summary, we have determined the best combination of RNA extraction and cell lysis method to obtain the maximum amount of RNA from fungal cells. In addition, we have established an efficient enrichment method that increases the proportion of fungal RNA without compromising gene expression. Together, these procedures provide a solution to comprehensively analyze transcriptomic profiles of C. albicans in samples biased towards human cells, such as clinical (vaginal) samples. Importantly, since the enrichment process is based on the differential lysis of human cells caused by the protective cell wall of the fungus, these procedures can be extended to other fungal pathogens. Furthermore, as the enrichment method uses centrifugation to separate human cell debris from intact fungal cells, the procedures can also be applied not only in transcriptomics but also in genomics studies.
Pages: 170 p.