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

seconds, and 72 C for 15 seconds. Fluorescence was monitored at the end of each 55 C incubation. The fluorescence detected in channel F2/F1 was analyzed using the LightCycler Analysis Software. The crossing point for each reaction was determined using the second derivative maximum algorithm and the arithmetic baseline adjustment. For quantification, a titration curve of neomycin mRNA was used.




Results and Discussion
Correlation of results obtained with and without amplification
Total RNA from Saccharomyces cerevisiae grown in rich or minimal medium was labeled with Cy3 and Cy5 by three different methods (Figure 1):
    100 g total RNA by reverse transcription (cDNA labeling/no amplification)
    10 g total RNA by linear amplification (cDNA synthesis followed by in vitro transcription)
    50 ng total RNA by random amplification (random PCR amplification followed by in vitro transcription).
Two hybridizations for each set of probes were performed on the MWG PAN YEAST Array, including a dye swap to minimize normalization and hybridization artifacts [7]. A threshold for low signal intensities was calculated based on the average signal intensities of 15 Arabidopsis-specific negative control spots. The average value of those 15 spots, plus the standard deviation multiplied by 1.96, gives a 95 % confidence that signals above this value are based on specific hybridization. Among a total of 6,250 spots, 75% (4,690) displayed signal intensities above the threshold. For those yeast ORFs, the average ratio of hybridization signals obtained from yeast grown in rich or minimal medium was determined. Within the 4,690 yeast ORFs, 562 ORFs displayed a ratio of ≥2, and 496 ORFs displayed a ratio o
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