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RNA Extraction Methods for Real-Time PCR and Microarray Analyses of Cryptosporidium and Toxoplasma gondii Oocysts - 2nd Presentation
Citation:
See, M. J., M. W. WARE, J. P. Dubey, AND E. VILLEGAS. RNA Extraction Methods for Real-Time PCR and Microarray Analyses of Cryptosporidium and Toxoplasma gondii Oocysts - 2nd Presentation. Presented at 10th International Workshop on Opportunistic Protists, Boxton, MA, May 28 - 31, 2008.
Impact/Purpose:
1) Refine new, practical methods for the detection of CCL-related and emerging waterborne human protozoa. 2) Evaluate new technologies for their use in method development. 3) Evaluate the efficacy of disinfection procedures on protozoans in collaboration with NRMRL scientists.
Description:
The ability of infectious oocyst forms of Toxoplasma gondii and Cryptosporidium spp. to resist disinfection treatments and cause disease may have significant public health implications. Currently, little is known about oocyst-specific factors involved during host cell invasion processes or about their survival after disinfection treatment. Microarrays and reverse transcriptase real-time PCR (RT-qPCR) are useful techniques that are amenable to identifying genes involved in regulating such mechanisms. In order to effectively perform these analyses, high quality RNA must be isolated. Here, we evaluated four RNA extraction methods (Ambion, RiboPure, Qiagen RNeasy, Epicentre Master Pure and TRIzol LS reagent) in their ability to extract high quality RNA from C. parvum and T. gondii oocysts. RNA quality was measured and expressed as an RNA Integrity Number (RIN), with a 9.0 or above indicating minimal degradation, while RNA purity was determined using RT-qPCR. Preliminary results indicated high quality RNA (RIN 8.3 – 9.8) was extracted from C. parvum oocysts using all four extraction methods; however, genomic DNA (gDNA) was present in all samples. DNase I treatment effectively removed gDNA contaminants only in Ambion and Qiagen extracted RNA, but the quality was compromised (RIN 5.5 - 7.7). RNA extracted from T. gondii oocysts proved to be more difficult and the quality/purity of RNA extracted was variable. Nevertheless, all RNA extraction methods used in this study were effective in quantitating hsp-70 and β-tubulin expression in C. parvum oocysts and SporoSAG expression in T. gondii oocysts using RT-qPCR. These studies will now enable us to use RT-qPCR, and perhaps microarray analysis, for identifying novel genes essential for oocyst development, survival in the environment, and the establishment of infection in the host.