Emilie Giraud, Evie Melanitou, Interactions Virus-Insectes - Insect-Virus Interactions (IVI), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], Département Parasites et Insectes vecteurs - Department of Parasites and Insect Vectors, Institut Pasteur [Paris], This work was supported by institutional funding from the Institut Pasteur., We are grateful to the members of the Immunophysiology and Parasitism Unit for their recommendations for these experimental procedures and their participation to the original work described in the published paper from where this protocol is derived (Giraud et al. al., 2019b, Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), and Institut Pasteur [Paris] (IP)
International audience; Parasites of the genus Leishmania infect the mammalian hosts, including mice and humans and cause cutaneous or visceral leishm aniasis depending upon the parasite species transmitted by the vector sandfly. Leishmania amazonensis is one of the Leishmania species responsible for th e cutaneous form of the disease. We have inoculated with these parasites the ear dermis of mice. RNA preparations were performed from fragmented tissues using a buffer containing guanidin isothiocynate (RLT buffer, RNeasy Mini Kit, Qiagen, SAS, France) and β mercaptoethanol. Both reagents facilitate the isolation of intact RNA from tissues and the use of the RNeasy Kits present with several advantages that facilitate the isolation of pure non degraded total RNA: i) T his method allows to avoid the presence o f phenol in the RNA extraction buffer, commonly used in alternative protocols; ii) Moreover Diethylpyrocarbonate (DEPC) treatment of glassware, to avoid RNAses contamination of the samples, is not required with this protocol; iii) F inally, it is a fast pro cedure and the isolated total RNA may be concentrated in a small volume thus facilitating its use for downstream experimental procedures.