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| Untersuchte Arbeit: Seite: 110, Zeilen: 1-17 |
Quelle: Bruder et al 2004 Seite(n): 628, 629, Zeilen: 628: r.col: last lines; 629: l.col: 1ff |
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| 1.11 DNA microarray hybridization and analysis
Total RNA from sorted AECII was isolated using the RNAeasy kit (Qiagen, Hilden, Germany). Quality and integrity of total RNA isolated from 2 x 105 sorted AECII cells was assessed by running all samples on an Agilent Technologies 2100 Bioanalyser (Agilent Technologies, Waldbronn, Germany). For RNA amplification the first round was done according to Affymetrix without biotinylated nucleotides using the Promega P1300 RiboMax Kit (Promega, Mannheim, Germany) for T7 amplification. For the second round of amplification the precipitated and purified RNA was converted to cDNA primed with random hexamers (Pharmacia, Freiburg, Germany). Second strand synthesis and probe amplification were done as in the first round with two exceptions: incubation with RNAse H preceeded the first strand synthesis to digest the aRNA, and the T7T23V oligo for initiation of the second strand synthesis was used. 12.5μg biotinylated cRNA preparation was fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix MG-U74Av2 chips for 16 hours. After hybridization, GeneChips were washed, stained with streptavidin-PE and read using an Affymetrix GeneChip fluidic station scanner. |
3.8 DNA microarray hybridization and analysis
The quality and integrity of the total RNA isolated from 5 x 104 cells was controlled by running all samples on an Agilent [page 629] Technologies 2100 Bioanalyzer (Agilent Technologies, Waldbronn, Germany). For RNA-amplification, the first round was done according to Affymetrix without biotinylated nucleotides using the Promega P1300 RiboMax Kit (Promega, Mannheim, Germany) for T7 amplification. For the second round of amplification the precipitated and cleaned aRNA was converted to cDNA using random hexamers (Pharmacia, Freiburg, Germany). Second-strand synthesis and probe amplification was done like in the first round, except for an incubation with RNAse H before first-strand synthesis to digest the aRNA and for the use of the T7T23V oligo for initiation of the synthesis of the second strand. The concentration of biotin-labeled cRNA was determined by UV absorbance. In all cases, 12.5μg of each biotinylated cRNA preparation was fragmented and placed in a hybridization cocktail containing four biotinylated hybridization controls (BioB, BioC, BioD, and Cre) as recommended by the manufacturer. Samples were hybridized to an identical lot of Affymetrix MOE430A for 16 h. After hybridization the GeneChips were washed, stained with SA–PE and read using an Affymetrix GeneChip fluidic station and scanner. |
Its just the description of material and methods, but the text is largely identical and the source is not given. |
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