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Pak. J. Bot., 48(2): 661-672, 2016.

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EFFICIENT RNA EXTRACTION PROTOCOL FOR THE WOOD MANGROVE SPECIES LAGUNCULARIA RACEMOSA SUITED FOR

NEXT-GENERATION RNA SEQUENCING

Mauricio Wolf Wilwerth¹, Priscilla de Barros Rossetto^1*, Fernanda Reinert², Raquel Soares Peixoto³ and Márcio Alves-Ferreira¹

Abstract: Mangrove flora and habitat have immeasurable importance in marine and coastal ecology as well as in the economy. Despite their importance, they are constantly threatened by oil spill accidents and environmental contamination; therefore, it is crucial to understand the changes in gene expression to better predict toxicity in these plants. Among the species of Atlantic coast mangrove (Americas and Africa), Laguncularia racemosa, or white mangrove, is a conspicuous species. The wide distribution of L. racemosa in areas where marine oil exploration is rapidly increasing make it a candidate mangrove species model to uncover the impact of oil spills at the molecular level with the use of massive transcriptome sequencing. However, for this purpose, the RNA extraction protocol should ensure low levels of contaminants and structure integrity. In this study, eight RNA extraction methods were tested and analysed using downstream applications. The InviTrap Spin Plant RNA Mini Kit performed best with regard to purity and integrity. Moreover, the obtained RNA was submitted to cDNA synthesis and RT-PCR, successfully generating amplification products of the expected size. These results show the applicability of the RNA obtained here for downstream methodologies, such as the construction of cDNA libraries for the Illumina Hi-seq platform.

Key words: L. racemosa; RNA isolation; RT-PCR; Mangrove; Guanidine (iso) thiocyanate.

¹Laboratório de Genética Molecular Vegetal, UFRJ, Av. Prof. Rodolpho Paulo Rocco, s/n, Bloco A CCS Cidade Universitária, 21941-617, Rio de Janeiro, RJ, Brazil

²Laboratório de Ecofisiologia Vegetal, UFRJ, Av. Prof. Rodolpho Paulo Rocco, s/n, Bloco A CCS Cidade Universitária, 21941-617, Rio de Janeiro, RJ, Brazil

³Laboratório de Ecologia Microbiana Molecular, UFRJ, Av. Prof. Rodolpho Paulo Rocco, s/n, Bloco K CCS Cidade Universitária, 21941-617, Rio de Janeiro, RJ, Brazil.

*Corresponding author’s email: MAF-marcioaf@ufrj.br; Phone - +55 21 39386381