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Gene Expression Arrays:,,,Highly Sensitive Detection of Expression Patterns,,,with Improved Tools for Target Amplification

on One microgram of Cy3- and Cy5-labeled cDNA or 10 g cRNA from the respective yeast population was used for hybridization. Hybridizations were performed overnight at 42 C using a buffer containing 50% formamide.

Yeast microarrays
Using a contact printing principle, 5-modified 40-bp oligonucleotides were covalently attached to the solid surface [6]. Each array was comprised of 6,250 oligonucleotides that detected different yeast open reading frames (ORFs).

Image analysis
Scanning for signal intensities was performed with a confocal laser scan microscope. Each slide was scanned six times per channel with an increasing photomultiplier setting to expand the dynamic range of the measurement. Resulting images were analyzed with the Imagene 4.0 Software; the output files were corrected between both channels via total signal intensities and local background subtraction using the MWG MAVI Software.

LightCycler RT-PCR
Quantitative RT-PCR was performed on the LightCycler Instrument using the DNA Hybridization Probe format. cDNA was produced from yeast total RNA in a 20-l reaction, including 20 g total RNA and a defined copy number of neomycin mRNA (spike-in control), oligo dT primer and avian myeloblastosis virus (AMV) reverse transcriptase. RNA and primers were first denaturated at 70 C for 10 minutes, then incubated at 42 C for 60 minutes, followed by a 5-minute denaturation at 94 C. Two hundred picograms and 20 pg of cDNA, primers and hybridization probes, and LightCycler FastStart DNA Master (as described in the protocol) were used in a 20-l reaction. The reaction conditions included: 10 minutes denaturation at 95 C, 45 cycles at 95 C for 10 seconds, 55 C for 15
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