Open AccessRapid methods to extract DNA and RNA from Cryptococcus neoformans
Author(s) -
Bolano Alessandro,
Stinchi Silvia,
Preziosi Roberta,
Bistoni Francesco,
Allegrucci Massimo,
Baldelli Franco,
Martini Alessandro,
Cardinali Gianluigi
Publication year - 2001
Publication title -
fems yeast research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.991
H-Index - 92
eISSN - 1567-1364
pISSN - 1567-1356
DOI - 10.1111/j.1567-1364.2001.tb00037.x
Subject(s) - cryptococcus neoformans , biology , rna , dna , nucleic acid , yeast , rna extraction , lysis , capsule , dna extraction , cryptococcus , extraction (chemistry) , microbiology and biotechnology , biochemistry , chromatography , polymerase chain reaction , gene , botany , chemistry
Extraction of nucleic acids from the pathogenic yeast Cryptococcus neoformans is normally hampered by a thick and resistant capsule, accounting for at least 70% of the whole cellular volume. This paper presents procedures based on mechanical cell breakage to extract DNA and RNA from C. neoformans and other capsulated species. The proposed system for DNA extraction involves capsule relaxation by means of a short urea treatment and bead beating. These two steps allow a consistent extraction even from strains resistant to other procedures. Yield and quality of DNA obtained with the proposed method were higher than those obtained with two earlier described methods. This protocol can be extended to every yeast species and particularly to those difficult to handle for the presence of a capsule. RNA purification is accomplished using an original lysing matrix and the FastPrep System (Bio101) after a preliminary bead beating treatment. Yields range around 1 mg RNA from 15 ml overnight culture (10 9 cells), RNA appears undegraded, making it suitable for molecular manipulations.