Resumo

SANDOVAL-PINEDA, Jhon Felipe; OCHOA-CORONA, Francisco Manuel  e  TORRES-ROJAS, Esperanza. Evaluation of different RNA extraction methods from the native fungus Xylaria sp. Rev. colomb. biotecnol [online]. 2017, vol.19, n.1, pp.42-54. ISSN 0123-3475.  https://doi.org/10.15446/rev.colomb.biote.v19n1.57114.

Obtaining high quality RNA is the first step for gene expression analysis. However, the low stability of this molecule and high presence of contaminants such as RNases, proteins and polysaccharides may trouble extractions. Fungi cell wall composition is highly diverse; therefore optimizing RNA extraction procedures is necessary when studying specific organisms. In this study, different methods of tissue homogenization (liquid nitrogen and lyophilization) and RNA extraction (Trizol, CTAB and RNeasy mini kit) were assessed with a native ascomycete, Xylaria sp. RNA purity, concentration and integrity were determined by spectrophotometry and electrophoresis. In addition, a set of housekeeping gene primers was designed targeting the β-tubulin gene. The primers were used to determine if the RNA extracted allowed RT-PCR amplification. It was demonstrated that homogenization of tissue by lyophilization allowed higher yields of RNA regardless of the extraction protocol used, however, the RNA integrity was affected. The higher RNA purity was obtained using CTAB and the higher yields using the RNeasy mini kit. The extracted RNA is amplifiable by RT-PCR regardless of the homogenization and extraction methodology used. However, it is recommended to homogenize the tissue with liquid nitrogen and to extract RNA with the RNeasy mini kit due the shortness and efficiency of these protocols.

Palavras-chave : Ascomycete; β-tubulin; lyophilization; liquid nitrogen; RT-PCR.

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