Total RNA Extraction from Granulomatous Lesions

Frozen granulomatous lesions were first macerated with the help of a pestle and pot. To proceed with total RNA extration, TRIzol LS (Thermo Scientific) was used according to the protocol described by Pisu et al., 2020 (link). For the control lung was used uninfected healthy lungs, while for the control yeasts was used the original yeast suspension used in the infection. The control lung and control fungus were mechanical lysed with pestle and pot and zirconium beads, respectively, followed by RNA extraction with TRIzol LS (Thermo Scientific) according to the manufacturer’s protocol. After RNA extraction, the DNA was digested with Rnase-free Dnase (RQ1 Rnase-Free Dnase, Promega Corporation) in the presence of the ribonuclease inhibitor Rnasin® (Promega Corporation). The purification was done with the addition of 1 volume of phenol:chloroform (1:1), followed by 100% ethanol:3M sodium acetate (10:1) RNA precipitation. RNA integrity was verified with a 2100 Bioanalyzer (Agilent) and RNA 6000 Nano Kit (Agilent).

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Borges B.M., Ramos R.B., Preite N.W., Kaminski V.D., Alves de Castro P., Camacho M., Maximo M.F., Fill T.P., Calich V.L., Traynor A.M., Sarikaya-Bayram Ö., Doyle S., Bayram Ö., de Campos C.B., Zelanis A., Goldman G.H, & Loures F.V. (2023). Transcriptional profiling of a fungal granuloma reveals a low metabolic activity of Paracoccidioides brasiliensis yeasts and an actively regulated host immune response. Frontiers in Cellular and Infection Microbiology, 13, 1268959.

Publication 2023

TRIzol LS RQ1 Rnase-Free Dnase Rnasin® 2100 Bioanalyzer RNA 6000 Nano Kit

Chloroform Dnase Ethanol Frozen Fungus Granulomatous lesions Infection Lung Phenol Promega Ribonuclease Rnase Sodium acetate Trizol Yeast Yeasts Zirconium

Corresponding Organization : Universidade Federal de São Paulo

Other organizations : Universidade de Ribeirão Preto, Universidade de São Paulo, Universidade Estadual de Campinas (UNICAMP), National University of Ireland, Maynooth

Top 5 similar protocols

Variable analysis

independent variables

Maceration of frozen granulomatous lesions with pestle and pot

dependent variables

Total RNA extraction

control variables

Uninfected healthy lungs used as control lung
Original yeast suspension used in the infection used as control yeast

controls

Positive control: Uninfected healthy lungs
Negative control: Original yeast suspension used in the infection

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