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Fungal RNA extraction and quantitative PCR.

SL Sophie Lev
KK Keren Kaufman-Francis
DD Desmarini Desmarini
PJ Pierre G. Juillard
CL Cecilia Li
SS Sebastian A. Stifter
CF Carl G. Feng
TS Tania C. Sorrell
GG Georges E. R. Grau
YB Yong-Sun Bahn
JD Julianne T. Djordjevic
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Fungal cells were pelleted by centrifugation and snap-frozen in liquid nitrogen. The cells were homogenized by bead beating in the presence of glass beads (425 µm to 600 µm) and TRIzol (Ambion), and RNA was extracted following the manufacturer’s instructions. Residual DNA in RNA samples was removed by treatment with RQ DNase (Promega). cDNA was synthesized using Moloney murine leukemia virus reverse transcriptase (Promega). Expression of PHO genes (listed in Table 1) was quantified by quantitative PCR (qPCR) on a Rotor-Gene 6000 (Corbett Research) using Platinum SYBR green qPCR SuperMix-UDG (Life Technologies, Inc.). Primers used for qPCR are listed in Table S1 in the supplemental material. The expression of each gene was quantified using the 2−ΔΔCT method, where actin (ACT1 [CNAG_00483]) served as a reference (housekeeping) gene.

Primers used in this study. Download TABLE S1, DOCX file, 0.01 MB.

Supplementary methods. Download TEXT S1, DOCX file, 0.02 MB.

Copyright and License information:  ©2017 Lev et al.
This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Creative Commons Attribution 4.0 International license.

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