The cultures were centrifuged (2 minutes, 8000 x g) and the supernatant was discarded. The pellet was immediately resuspended in 1.5 ml RNAprotect Bacteria Reagent (Qiagen, Hilden, Germany) to minimize RNA degradation.
Growth protocol
Prior use, the strain was stored in cryovials at -80°C (Cryobank; Mast Diagnostica, Bootle, England). For initial growth, cells were grown using a rotary shaker (Unimax 1010 and Incubator 1000; Heidolph, Schwabach, Germany) in alkaline peptone water (APW; 0.3% Yeast-Extract, 1% Peptone, 2% NaCl; pH 8.6) at 37°C overnight.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated using the peqGold Bacterial RNA Kit (Peqlab, Erlangen, Germany). Obtained RNA was eluted into 43 µl of DEPC-treated, DNase- and RNase-free water (Carl Roth, Karlsruhe, Germany). Samples were then treated by DNase I along with an RNase-A, -B and -C inhibitor (Fermentas, Vilnius, Lithuania). RNA quantity was measured by spectrophotometry (NanoDrop 2000, Thermo Scientific). RNA quality of each sample was monitored via gel electrophoresis. Additionally, the RNA quality was assessed using the Agilent RNA 6000 Nano Kit on a 2100 Bioanalyzer (Agilent, Santa Clara, US).
Label
Cy3
Label protocol
Reverse transcription of the RNA was performed. Briefly 200 ng of RNA were linear amplified using the full spectrum MultiStart primer (Biocat, Heidelberg, Germany) and Moloney murine leukemia virus (MMLV) reverse transcriptase (Agilent). The amplification was performed at 40°C for 2 hours followed by 65°C for 15 minutes and stored at 4°C. Labelling was performed using the Quick Amp Labeling Kit (Agilent). Briefly the amplified cDNA, the full spectrum MultiStart primer and T7 RNA polymerase were used along with Cyanine 3-CTP. Labeled cRNA was purified using the Qiagen RNeasy Mini Kit (Qiagen). A 3 µl-aliquot was used for quality control (NanoDrop 2000, Thermo Scientific).
Hybridization protocol
Finally 600 ng of the labelled and linear amplified cRNA (specific activity 9,0 pmol Cy3/µg cRNA) was fragmented, added to 25 µl Agilent-hybridization buffer resulting in 480 ng loaded on a microarray in a hybridization chamber (following manufacturers instructions). The hybridization was performed at 65°C for 17 hours and 10 rpm. Washing of the slides was performed using preheated washing buffer (Gene expression wash buffer kit, Agilent). First the chamber was rinsed with washing buffer. Then the slides were washed once followed by a second wash using washing buffer containing 0.01% Triton X-102 (Agilent). The slides were dried using acetonitrile.
Scan protocol
Scanning was carried out immediately after washing using the Agilent G2565CA scanner with a resolution of 5 µm.
Description
Gene expression after incubation at 37°C for 30min. Vp-37-w1
Data processing
After scanning, tiff-files were analysed and raw data was extracted using Feature Extraction Software (Agilent). Data processing was performed using Bioconductor V 2.12 package of the software R. At first, filtering of bad quality spots was performed and the data was background corrected with normexp method using offset = 50.
