( ssful PCR amplifications wer...)Amplification of Mouse cDNAs for Microarrays Using the Eppendorf ,,, MasterTaq Kit

ssful PCR amplifications were verified by electrophoresis on agarose gels. PCR products were purified on a BioRobot 3000 (QIAGEN, Santa Clarita, CA) using the QIAquick 96 PCR BioRobot Kit. The yield of PCR amplicon was assayed using the Picogreen dsDNA quantitation kit (Molecular Probes) and a Bio-Tek FLX800 microplate fluorescence reader.

Preparation of fluorescently labeled cDNA
One g of polyA+ RNA (mouse spleen and liver, respectively) were converted into fluorescently labeled cDNA by incubation with an oligo dT primer (44 M final) at 70C for 10 minutes in a final volume of 9 l. The solution was then mixed with 11 l of a solution 1.8X first strand buffer, containing 18 mM DTT, 900 M dATP, 900 M dGTP, 900 M dTTP, 230 M dCTP, 230 M Cy3- or Cy5-dCTP (APBiotech, UK), 20 U of RNasin (Promega), and 200 U Superscript II reverse transcriptase (Life Technologies). The reaction was incubated at 42C for 2 hours and heated at 100C for 5 minutes. RNA was hydrolyzed by the addition of 1 l of 10 N NaOH and subsequent incubation at 37C for 15 minutes. The reaction was neutralized by the addition of 5 l of 1M Tris pH 7.2 and 2 l of 5N HCl, and cDNA probes were purified using a QIAquick PCR purification kit (Qiagen, Santa Clarita, CA). Typically, one-third of each reaction was used per hybridization.

Microarray fabrication, hybridization, washing, scanning, and data analysis
PCR products (1,152 in total) derived from the mouse Unigene set and control cDNA clones were printed on a reflective slide (APBiotech, UK). DNA clones were heat denatured for 3 minutes at 95C, chilled on ice, and arrayed using a Molecular Dynamics (Sunnyvale, CA) Generation III spotter.
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