A method for extracting a high-quality RNA from Symbiodinium sp.
- 814 Downloads
Good quality RNA is essential for a range of analyses including microarray and gene expression studies. A number of methods for RNA extraction from symbiotic dinoflagellates were assessed for their ability to recover a high-quality RNA applicable for evaluation of gene expression profiles. The recovery and quality of the obtained RNA were evaluated with respect to UV light absorbance profiles and automated microcapillary electrophoretic RNA separation. A modified RNA extraction procedure that combines two existing commercial kits, Trizol and Qiagen RNeasy kits, was efficiently employed for the recovery of a high-quality RNA under specific homogenization conditions. Cell homogenization using glass beads at the speed of 4,500 rpm for up to 6 min resulted in a good RNA recovery and preserved RNA integrity. A high-quality RNA obtained following the described procedure was successfully applied in reverse transcriptase-polymerase chain reaction (PCR) and in quantitative PCR studies. Gene expression profiles were changed with RNA extraction procedure, with the highest transcript numbers at precise conditions of cell homogenization. RNA samples with RNA integrity number values from 6 and above were recommended for downstream applications. This sequence of RNA isolation and RNA evaluation represents a methodological improvement required for functional genomic studies in dinoflagellates.
KeywordsDinoflagellate Symbiodinium RNA Real-time PCR Gene expression Bioanalyzer
The authors would like to thank Dr. Rebecca Hawthorne for her assistance in using the Agilent 2100 Bioanalyzer and Drs. Iris Depaz and Shona Osborne for their help in the revision of the manuscript. This work was supported by ARC Centre of Excellence for Coral Reef Studies.
- Copois V, Bibeau F, Bascoul-Mollevi C, Salvetat N, Chalbos P, Bareil C, Candeil L, Fraslon C, Conseiller E, Granci V, Maziere P, Kramar A, Ychou M, Pau B, Martineau P, Molina F, Del Rio M (2007) Impact of RNA degradation on gene expression profiles: assessment of different methods to reliably determine RNA quality. J Biotechnol 127:549–559. doi: 10.1016/j.jbiotec.2006.07.032 CrossRefPubMedGoogle Scholar
- Imbeaud S, Graudens E, Boulanger V, Barlet X, Zaborski P, Eveno E, Mueller O, Schroeder A, Auffray C (2005) Towards standardization of RNA quality assessment using user-independent classifiers of microcapillary electrophoresis traces. Nucleic Acids Res 33:e56. doi: 10.1093/nar/gni054 CrossRefPubMedGoogle Scholar
- Sambrook J, Fritsch E, Maniatis T (1989) Molecular cloning: a laboratory manual. Cold Spring Harbor, New YorkGoogle Scholar
- Takishita K, Ishida K, Maruyama T (2003) An enigmatic GAPDH gene in the symbiotic dinoflagellate genus Symbiodinium and its related species (the order Suessiales): possible lateral gene transfer between two eukaryotic algae, dinoflagellate and euglenophyte. Protist 154:443–454. doi: 10.1078/143446103322454176 CrossRefPubMedGoogle Scholar
- Vesk M, Hallegraeff GM, Jeffrey SW (1990) Biology of marine plants. In: Clayton MN, King RJ (eds) Biology of marine plants. Longman Cheshire, Melbourne, pp 133–148Google Scholar