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The design employed microarray probes optimized for uniformity, sensitivity, and specificity, with probe properties computed using the latest thermodynamic models.
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First, microarray probes were optimized by screening 244,000 probes for hybridization with RNA from infected and uninfected macrophages.
Microarray probes were matched to gene identifiers using the CDF array annotation (version 18) provided by the University of Michigan microarray© lab56.
With this method, microarray probes with confusing nomenclature and quality differences can be identified.
Fig. 3 Transcripts identified in RAP annotations and 44 K microarray probes.
In both root and shoot samples, about 95% of transcripts were supported by the microarray probes.
The target gene regions of microarray probes were retrieved from the annotation file for human U133 Plus 2.0 (http://www.affymetrix.com).
Microarray probes were designed primarily from P. taeda hybridised with cRNA constructed from the close relative P. radiata.
Heterologous hybridisation requires microarray probes that are capable of tolerating a certain degree of mismatch with the target transcripts.
A comprehensive consensus sequence collection of Pinus cDNA sequences was collated into a non-redundant database used for automated design of 60-mer oligonucleotide microarray probes.
The differential expression of transcripts (treated vs. untreated) was determined from unfiltered microarray probes using the Benjamini-Hochberg method with adjusted p < 0.01 [28].
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