Malays. Appl. Biol. (2014) 43(2): 71-79
COMPARISON OF RNA EXTRACTION METHODS FOR TRANSCRIPT ANALYSIS FROM THE PSYCHROPHILIC YEAST, Glaciozyma antarctica
Bharudin, I.1, Zaki. N.Z.1, Abu Bakar, F.D.1, Mahadi, N.M.2, Najimudin, N.3, Illias, R.M.4 and Murad, A.M.A.*1
1School of Biosciences and Biotechnology, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor, Malaysia.
2Malaysia Genome Institute, Jalan Bangi, 43000 Kajang, Selangor, Malaysia.
3School of Biological Sciences, Universiti Sains Malaysia, 11800 Penang. Malaysia.
4Department of Bioprocess Engineering, Faculty of Chemical Engineering, Universiti Teknologi Malaysia, 81310 Skudai, Johor, Malaysia.
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ABSTRACT
To survive in extremely cold environments, psychrophilic microorganisms produce exopolysaccharides (EPS), which are carbohydrate polymers that constitute a substantial component of the extracellular polymers surrounding cells of these microorganisms. EPS can interfere with RNA extraction and decrease the purity of the RNA extracted from EPS producing microorganisms. In this work, six commercial RNA extraction kits and two published protocols for RNA extraction were evaluated for total RNA extraction from the psychrophilic yeast, Glaciozyma antarctica. All the protocols were optimised to obtain the highest quality of total RNA. The results show that all of the tested commercial kits and the tested conventional methods yielded RNA from G. antarctica, albeit with varying quality. The protocol that utilizes TRIzol® reagent was the most effective method for isolating total RNA from G. antarctica of which this protocol resulted in the highest RNA yield and purity compared to other methods. This method of RNA extraction produced RNA of sufficient quality for reverse transcriptase PCR (RT-PCR) to detect the expression of the G. antarctica delta 9-fatty acid desaturase gene as well as for the construction of a G. antarctica cDNA library.
Keywords: Glaciozyma antarctica, RNA extraction, cDNA library, psychrophiles
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