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RNA Extraction Methods for Reverse Transcriptase Real-Time PCR and Microarray Analysis of Cryptosporidium and Toxoplasma gondii Oocysts
Citation:
See, M. J., M. W. WARE, J. P. Dubey, AND E. VILLEGAS. RNA Extraction Methods for Reverse Transcriptase Real-Time PCR and Microarray Analysis of Cryptosporidium and Toxoplasma gondii Oocysts. Presented at ASM Annual Meeting, Muncie, IN, March 28 - 29, 2008.
Impact/Purpose:
This task focuses on the development, evaluation, and standardization of innovative methods, technologies, and procedures to determine the parasite burden of source and drinking water. Information as to the presence of these organisms in water supplies may assist communities to make informed decisions concerning their public health and infrastructure. Objective: 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. This work in this task supports CCL2 and 3 and is expected to be completed by 9/07.
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 establishment of infection in the host.