Protocol 42 Microarray experiments

The cDNA microarrays encompassed 1794 clones for oncologically relevant genes and 2314 genes and expressed sequence tags (ESTs) found to be differentially expressed in previous work (19). With further control genes, altogether the microarrays contained 4207 genes and ESTs. Insert DNA was amplified from bacterial clones by PCR using vector-specific primers. The PCR products were precipitated by isopropanol, washed in 70% ethanol, dried and dissolved in spotting buffer consisting of 3 x SSC/1.5 M betaine. Presence of product bands was confirmed by agarose gel electrophoresis (Ready-to-Run, Amersham Pharmacia Biotech, Freiburg, Germany). The DNA was spotted in duplicate onto epoxysilane-coated glass slides (Quantifoil, Jena, Germany) using the Omnigrid (Genemachines, San Carlos, CA) arrayer and SMP3 split pins (Telechem, Sunnyvale, CA). After spotting, microarrays were rehydrated, and DNA was denatured with boiling water prior to washing with 0.2% SDS, water, ethanol, and isopropanol. The arrays were dried with pressurized air.

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