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Evaluation of Four RNA Extraction Methods for Gene Expression Analyses of Cryptosporidium parvum and Toxoplasma gondii Oocys
Citation:
SEE, M. J., S. E. Staggs, J. P. Dubey, AND E. VILLEGAS. Evaluation of Four RNA Extraction Methods for Gene Expression Analyses of Cryptosporidium parvum and Toxoplasma gondii Oocys. JOURNAL OF MICROBIOLOGICAL METHODS. Elsevier Science Ltd, New York, NY, 89(3):185-192, (2012).
Impact/Purpose:
The overall objective of this task is the development of improved occurrence detection methods for protozoan parasites and Microsporidia. Since this work is a primary focus of the Branch, this task supports several individual projects related to sample preparation and protozoan detection. Together these projects will lead to complete methods able to support the UCMR and CCL3.
Description:
Cryptosporidium spp. and Toxoplasma gondii are important coccidian parasites that have caused waterborne and foodborne disease outbreaks worldwide. Techniques like subtractive hybridization, microarrays, and quantitative reverse transcriptase real-time polymerase chain reaction (RT-qPCR) assays have been used to understand the roles of specific genes in regulating life stage development and pathogenesis of these parasites. Key to the success of these approaches is isolating high quality messenger RNA (mRNA), which is particularly difficult with coccidian oocysts. Although commercial kits can provide high quality mRNA to study gene expressions in mammalian cells, their performances have not been thoroughly evaluated on oocysts. In this study, four RNA extraction kits: RiboPure-bacteria, MasterPure RNA, RNeasy micro, and TRIzol LS reagent kits were evaluated for their ability to isolate high quality mRNA. Results revealed that all four kits easily isolated total RNA from C. parvum oocysts. Analysis of total RNA quality as measured by RNA integrity number (RIN) showed sufficiently high quality values ranging from 8.4 to 9.8. However, genomic DNA (gDNA) contamination was present in all extracts. Additional DNase I treatment effectively removed gDNA contaminants, but partially degraded the RNA (RIN = 5.0 – 7.7). Total RNA isolations from T. gondii oocysts were also attempted and were partially successful, yielding RNA extracts sufficient for only RTqPCR. Overall, the RNeasy micro kit with additional DNase I treatment was the most effective for extracting sufficiently high quality total RNA from C. parvum and T. gondii oocysts.