Gene Expression Analysis of Heart Tissue
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Cross-sections of mouse ventricle (50 mg) were frozen at-90°C. To purify poly-A mRNA, frozen heart tissue was placed in OL1 buffer (Qiagen, Valencia, CA) with 3% b-mercaptoethanol(bME), homogenized with a tissue tearer (Biospec, Bartlesville, OK), passed through a shredder column (Qiagen), separated from protein, resuspended with Oligotex (Qiagen), eluted, precipitated with ethanol, dried in a vacuum, and resuspended in dH2O. Next, double-stranded cDNA was generated from the purified poly-A mRNA (SuperScript Kit, GibcoBRL, Grand Island, NY). The cDNA was subjected to in vitro transcriptionto form cRNA (Megascript Kit, Ambion, Austin, TX), incorporating biotinylatedCTP and UTP nucleotides (Enzo Diagnostics, Farmingdale, NY).
Biotinylated mouse heart cRNA (10 mg) was fragmented into lengths of 50-100 nucleotides (Mg2+ buffer, 94°C, 35 min)and mixed with a hybridization cocktail (bacterial control cRNAs for BioB,C, D, and Cre; control 948B biotinylated oligo [Affymetrix Inc., Santa Clara,CA.]; herring sperm DNA, and 10 mM Tris-HCl, pH 7.6, 1 M NaCl, and 0.01%TritonX-100). The cocktail was hybridized to the DNA probe array for 18-24hr at 45°C at 60 rpm. The DNA array was then rinsed 10 times, washed in hybridization buffer (0.1x) for 20 min at 50°C at 60 rpm, and stained with a streptavidin-phycoerythrin solution (25 mg/ml, hybridization buffer1.0x, 1 mg/ml acetylated bovine serum albumin at room temperature at 60rpm in the dark). The DNA array was washed another 10 times.
DNA arrays were excited by an argon-ion laser; the emission was detected by a photomultiplier tube and digitized after confocal scanning. GeneChip 3.0 automated analysis software (Affymetrix Inc., Santa Clara,CA) was used to collect hybridization patterns and intensity information for each sample and to analyze the data. The hybridization intensities were scaled by setting the total fluorescence intensity of a DNA array to a fixed value of 500,000 (gene expression was calculated in absolute hybridization intensity units).
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