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Open Access Highly Accessed Methodology article

Analysis of current and alternative phenol based RNA extraction methodologies for cyanobacteria

Fernando Lopes Pinto, Anders Thapper, Wolfgang Sontheim and Peter Lindblad*

Author Affiliations

Department of Photochemistry and Molecular Science, The Ångström Laboratories, Uppsala University, Box 523, SE-75120, Uppsala, Sweden

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BMC Molecular Biology 2009, 10:79  doi:10.1186/1471-2199-10-79

Published: 7 August 2009

Abstract

Background

The validity and reproducibility of gene expression studies depend on the quality of extracted RNA and the degree of genomic DNA contamination. Cyanobacteria are gram-negative prokaryotes that synthesize chlorophyll a and carry out photosynthetic water oxidation. These organisms possess an extended array of secondary metabolites that impair cell lysis, presenting particular challenges when it comes to nucleic acid isolation. Therefore, we used the NHM5 strain of Nostoc punctiforme ATCC 29133 to compare and improve existing phenol based chemistry and procedures for RNA extraction.

Results

With this work we identify and explore strategies for improved and lower cost high quality RNA isolation from cyanobacteria. All the methods studied are suitable for RNA isolation and its use for downstream applications. We analyse different Trizol based protocols, introduce procedural changes and describe an alternative RNA extraction solution.

Conclusion

It was possible to improve purity of isolated RNA by modifying protocol procedures. Further improvements, both in RNA purity and experimental cost, were achieved by using a new extraction solution, PGTX.