genomewide characterization of host-pathogen interactions by ...

Maren Depke
Material and Methods
Pathogen Gene Expression Profiling
RNA preparation
RNA was prepared using the acid-phenol-method. The lysates / aqueous phases were
extracted twice with an equal volume of acid phenol solution (Roth, Karlsruhe, Germany) and
once with an equal volume of chloroform / isoamyl alcohol (3-methyl-1-butanol) mix (24 vol. of
chloroform and 1 vol. of isoamyl alcohol equilibrated with 1 M Tris/HCl pH 8.0). RNA was
precipitated from the remaining cleaned aqueous phase with 1/10 volume of 3 M sodium acetate
pH 5.5 and 1 volume of isopropanol (2-propanol) overnight at −20°C. After two washes with
−20°C pre-cooled 80 % ethanol, the RNA was dried at room temperature and solved in nucleasefree
water (Ambion Inc., Austin, TX, USA, now part of Applied Biosystems, Foster City, CA, USA).
To avoid the influence of potential DNA contamination, the RNA was DNase treated and
afterwards purified using the RNA Clean-Up and Concentration Kit (Norgen Biotek Corp., Thorold,
ON, Canada; distributed by BioCat GmbH, Heidelberg, Germany). The concentration was
determined photometrically (NanoDrop ND-1000, NanoDrop Technologies, Wilmington, DE,
USA), and the quality was checked with an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa
Clara, CA, USA).
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