genomewide characterization of host-pathogen interactions by ...

Maren Depke
Material and Methods
Kidney Gene Expression Pattern in an in vivo Infection Model
RNA preparation
A frozen aliquot of disrupted tissue was disintegrated mechanically at 2600 rpm for 2 min in a
bead mill (Mikrodismembrator S, B. Braun Biotech International GmbH, Melsungen, Germany;
now part of Sartorius AG, Göttingen, Germany) with 0.5 ml TRIZOL (Invitrogen, Karlsruhe,
Germany) using liquid nitrogen cooled Teflon vessels. Thereafter, another 0.5 ml TRIZOL was
added to the still frozen lysate. After thawing, the lysate was incubated for 10 min at room
temperature, flash-frozen in liquid nitrogen and stored −70°C until RNA preparation was
continued. The lysates were again thawed at room temperature. Chloroform was added (200 µl
chloroform / 1 ml TRIZOL), samples were shaken vigorously for 15 s and incubated at room
temperature for 5 min. Organic and aqueous phase were separated by centrifugation (12000 x g,
15 min, 4°C) and RNA was precipitated from the aqueous phase with 500 µl isopropanol (2-
propanol) / 1 ml TRIZOL overnight at −20°C. After two washes with −20°C pre-cooled 80 % ethanol
RNA was dried at room temperature and dissolved in nuclease-free water (Ambion Inc., Austin,
TX, USA, now part of Applied Biosystems, Foster City, CA, USA).
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).
DNA array analysis
The RNA expression profile was analyzed on GeneChip Mouse Gene 1.0 ST arrays (Affymetrix,
Santa Clara, CA, USA) using the Whole Transcript (WT) Sense Target Labeling and Control
reagents according to the manufacturer’s instructions. Arrays were washed and stained in a
GeneChip FluidicsStation 450 and scanned with a GeneChip Scanner 3000 (all: Affymetrix).
DNA array data analysis
The array image files (CEL) were first quality controlled in the Expression Console software
(Affymetrix) and then imported into the Rosetta Resolver software (Rosetta Biosoftware, Seattle,
WA, USA) for data analysis. Signals were generated and normalized using the RMA algorithm.
Groups were compared in log-transformed space using error-weighted one-way ANOVA with
Benjamini-Hochberg False Discovery Rate multiple testing correction and p* < 0.01 was regarded
as significant. The control sequences (e. g. negative, positive, polyA, and hybridization controls)
and sequences that were not expressed on all arrays in the selected group comparison (with
p > 0.01 on intensity profile level in Rosetta Resolver software) were not included in statistical
testing. Afterwards, sequence sets were translated into EntrezGene records using the Rosetta
Resolver annotation of December 2009. Two criteria were used for definition of differential
expression: Significance in ANOVA statistical testing and a minimal absolute fold change of 2.
Genes significant in ANOVA but with a minimal absolute fold change of only 1.5 were considered
to be regulated by trend.
Ingenuity Pathway Analysis (IPA) of gene expression data
EntrezGene identifiers and fold change gene expression data of differentially expressed genes
were imported to the Ingenuity Pathway Analysis tool (Ingenuity Systems Inc.,
www.ingenuity.com) and analyzed using all genes of the GeneChip Mouse Gene 1.0 ST array as
reference set without further restriction.
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