Master of Science in Chemical Engineering (MSChE)
Research on production of polyhydroxyalkanoates (PHA) by microorganisms has captured attention over the last two decades, since PHA have similar properties as petroleum-based thermoplastics, yet are close to carbon neutral and made from renewable sources. Cyanobacteria are considered to be good PHA producers because of their simple nutrient requirements (mainly water, sunlight and CO2) and fast cell growth. However, knowledge of metabolisms behind PHA production by cyanobacteria is limited. Therefore, gene expression analysis of cyanobacteria is required to get a thorough understanding at the molecular level, and gene expression analysis requires the extraction of high quality RNA. Extracting high quality intact RNA from cyanobacteria is problematic due to their complicated cell wall structure and excessive polysaccharides produced by cells. Previous work on RNA extraction from cyanobacteria is either strain-specific or involves handling of hazardous chemicals like toxic chaotropic agents, saturated hot phenol or liquid nitrogen. With this work, we developed a low-cost RNA extraction method using a simple CTAB extraction buffer. Three morphologically distinct cyanobacteria strains, Synechocystis sp. PCC 6803, Plectonema sp. strain UTEX 1541, and Nostoc muscorum UTEX 1037 were used to testify the validity of this new method. Two traditional extraction protocols, using the Qiagen RNeasy Mini kit and TRIzol Reagent respectively for cyanobacteria were also optimized and analyzed with the same species. The newly developed CTAB method successfully extracted total RNA of high quality and quantity from the three selected strains, and the extracted total RNA were of sufficient quantity and quality for RT-PCR after DNase I treatment. Compared to the two traditional extraction methods, both the purity and the yield of extracted total RNA were greatly improved when using CTAB method: yields was improved up to 13 times higher, and both the A260/A280 and A260/A230 ratio indicated less contaminations in extracted RNA. Furthermore, the experimental cost of CTAB method was significantly lowered by up to 83%, yet still easy to perform.
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Zhang, Zenghui, "A novel RNA extraction method for cyanobacteria" (2012). LSU Master's Theses. 3899.