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United States Department of Agriculture

Agricultural Research Service

Research Project: INTEGRATED STRATEGIES FOR ADVANCE MANAGEMENT OF FRUIT, NUT, AND OAK TREE DISEASES

Location: Crops Pathology and Genetics Research

Title: Evaluation of automated cell disruptor methods for oomycetous and ascomycetous model organisms

Authors
item Kasuga, Takao
item Bui, Mai

Submitted to: Fungal Genetics Reports
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: June 14, 2011
Publication Date: October 1, 2011
Repository URL: http://www.fgsc.net/FGR/FGR58/FGR58Kasuga.htm
Citation: Kasuga, T., Bui, M.Q. 2011. Evaluation of automated cell disruptor methods for oomycetous and ascomycetous model organisms. Fungal Genetics Reports. 58:4-13.

Interpretive Summary: High-throughput RNA extraction methods for a plant pathogenic oomycete Phytophthora capsici were developed and qualities of obtained RNA samples were evaluated by means of microarray global mRNA profiling. We found that RNA expression noise can be introduced during high-throughput RNA extractions. In this report we discuss the nature of the noise and means to reduce it.

Technical Abstract: Two automated cell disruptor-based methods for RNA extraction; disruption of thawed cells submerged in TRIzol Reagent (method QP), and direct disruption of frozen cells on dry ice (method CP), were optimized for a model oomycete, Phytophthora capsici, and compared with grinding in a mortar and pestle under liquid nitrogen (method MP). The above three methods resulted in reproducible global mRNA profiles according to microarray mRNA profiling. It was, however found that when frozen samples were allowed to thaw at 4°C prior to the addition of TRIzol Reagent in method QP, which was a possible incident during a prolonged high-throughput application and also was a part of the procedure in the wildly-used hot phenol protocol, global mRNA profiles were altered significantly. These thawed samples, however, retained high RNA integrity, thus were able to pass conventional stringent quality control measures. This demonstrated the necessity of microarray mRNA profiling for the evaluation of RNA extraction protocols. Although P. capsici did not survive the freeze-thaw process, it is implied that thawed samples were able to transcriptionally respond to cryoinjury. In order to minimize the noise associated with RNA extraction, we advocate the use of frozen-phase cell disruption methods such as CP and MP.

Last Modified: 8/22/2014
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