Total RNA was isolated using RNAprotectTM bacteria reagent (Qiagen) and an RNeasy kit (Qiagen) according to the manufacturer’s protocol with an optional step of on-column DNase treatment with RNase-Free DNase (Qiagen). RNA concentration was measured with a NanoDrop ND-1000 (NanoDrop Technologies, Wilmington, DE)
Label
cy5
Label protocol
cDNA was synthesized using aminoallyl-dNTP according to the manufacturer’s instruction. After purification, aminoallyl labeled cDNA was fluorescently labeled with Cy-3/cy-5 mono-Reactive Dye Pack (Amersham Biosciences, Piscataway, NJ)
Hybridization protocol
equal amounts of labeled cDNA of the wild-type and the mutant were mixed and dried in a speed vacuum concentrator. After resuspension with hybridization buffer provided by the slide manufacturer, the mixture was denatured by heating at 95 ◦C for 10 min. The hybridization mixture was loaded onto a microarray slide and hybridized in a shaking incubator at 42 ºC for 24 h. Slides were washed as the manufacture’s recommendations and dried by centrifugation.
Scan protocol
Using a ScanArray Express laser scanner (Applied Biosystem Inc., Foster City, CA) each dye channel of each slide was scanned at a 10 μm resolution using three predetermined laser power and photomultiplier gain settings. Thus for each slide six scans were performed, three separate scans at different settings of each of the two dye channels.
Description
none
Data processing
ImaGene software (BioDiscovery, El Segundo, CA) was used for spot finding and to measure the spot-specific mean signal strength of each spot. The fluorescentc intensities of all spots were log2 transformed. The median of the three log2-transformed spot signals was determined and this value was used as spot signal for all further calculations. As an additional measure against intensity dependent dye bias the data was then subjected to LOWESS normalization.
