Alessandro Bolano, Silvia Stinchi, Roberta Preziosi, Francesco Bistoni, Massimo Allegrucci, Franco Baldelli, Alessandro Martini, Gianluigi Cardinali.
FEMS Yeast Research. 2001. Vol 1.

Introduction

Extraction of nucleic acids from the pathogenic yeast Cryptococcus neoformans is hampered by a thick and resistant capsule accounting for at least 70% of the whole cellular volume.

This study presents an effective procedure based on mechanical cell breakage using the FastPrep® system to extract RNA from
C. neoformans and other capsulated species.

Overview

  • Keywords: Yeast, DNA, RNA, Extraction, Capsule, Method, Cryptococcus neoformans
  • Aim of the study: Development of consistent extraction method for DNA & RNA extraction from resistant strains of Cryptococcus neoformans
  • Application: RNA extraction
  • Sample name: Cryptococcus neoformans
  • Sample type: Yeast
  • Material: FastPrep-24™ instrument, 0.4-0.6 mm glass beads
  • Buffers: TE buffer, RNA lysing solution (0.5% SDS and 0.5% N-laurylsarcisine)

Protocols & Parameters

Cells were grown in YEPD (yeast extract 1%, peptone 1%, dextrose 2%) for 18h at 25°C in 500-ml shaken (150 rpm) flasks witha liquid to air ration of 1:10.
  1. Cells from 15 ml overnight culture (approx.109) were collected, washed with cold water, resuspended in 200 μl of TE
    and distributed into two 1.7 ml microcentrifuge tubes.
  2. 0.5 ml of glass beads (0.4-0.6 mm), 250 μl of RNA lysing solution, 250 μl 4 M guanidine thiocyanate with 25 mM
    sodium citrate, pH 7, 0.1 M β-mercaptoethanol, 500 μl phenol, pH 5 and 100μl chloroform/isoamyl alcohol (24 :1) were added
    to each tube.
  3. Both tubes were placed in the FastPrep-24™ instrument and processed in 4 cycles of 40 sec each. Between the cycles
    samples were placed on ice.
  4. After last cycle tubes were removed from the instrument, placed 5 min in ice and spun 10 min at 13,000 x g.
  5. The upper phase was transferred to fresh microcentrifuge tube for RNA purification.

Results

Effective C. neoformans RNA extraction from clinical isolates

Electrophoresis of total cellular RNA extracted from
C. neoformans and S.cerevisiae on a 0.8% agarose.

Lane M, 1 kb ladder. Lanes a, b, c and d, C. neoformans RNA extracted from clinical isolates from cerebrospinal fluid of an AIDS patient (a, first episode; b, relapse) and from CBS collection (c, CBS 6995 encapsulated strain; d, CBS 7698 non-capsulated strain). Lane e, S. cerevisiae RNA extracted from the DBVPG 6820 strain.

Conclusion

  • RNA purification is accomplished using FastPrep® system and glass beads after a preliminary bead beating treatment
  • Yields range around 1 mg RNA from 15 ml overnight culture (109 cells)
  • RNA appears undegraded, making it suitable for molecular manipulations