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URL: https://dx.doi.org/10.17504/protocols.io.wqdfds6

Authors: Sebastian Najle

Group: Multicellgenomelab

Summary: This protocol describes the steps for isolation of total RNA for cDNA synthesis in the marine Ichthyosporean Sphaeroforma arctica. In general, Ichthysoporeans are characterized by having a thick cell wall, of uncknown composition, that is particularly hard to brake, making cell lysis difficult. Here we have introduce a simple modification on the conventional RNA extraction protocol using TRIzol reagent, by including two cycles of freezing-tawing of the cells-TRIzol suspension, in order to facilitate cell lysis. This modification improved significantly the RNA isolation yield. Total RNA is further treated with DNAse I enzyme and purified by precipitation to minimize genomic DNA contamination. Highly pure total RNA is then used to synthetize cDNA for downstream applications. .justify:after { content: ""; display:inline-block; width: 100%; } .justify:after { content: ""; display:inline-block; width: 100%; }

Associated Publications: Ocaña-Pallarès E, Najle SR, Scazzocchio C, Ruiz-Trillo I (2019) Reticulate evolution in eukaryotes: Origin and evolution of the nitrate assimilation pathway. PLoS Genet 15(2): e1007986. doi: 10.1371/journal.pgen.1007986

Affiliations: Institut de Biologia Evolutiva (CSIC-Universitat Pompeu Fabra)

External URL: https://doi.org/10.1371/journal.pgen.1007986

Version: 1

Publication Date: 2019

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