Gender: male Age: 42 Tissue: adrenal gland Cause of death: acute cardiovascular failure
Extracted molecule
genomic DNA
Extraction protocol
genomic DNA isolation with phenol extraction according to standard protocol (Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular cloning; a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989)
Label
Cy5
Label protocol
Labeling was done using 2 mcg DNA, according to the BioPrime® Array CGH Genomic Labeling System protocol supplied by the manufacturer (Invitrogen, Carlsbad, CA). We used CY3 dCTP and CY5 dCTP respectively (PA53021 and PA55021, GE Healthcare, Piscataway, NJ). The labeled DNA probes were mixed together and combined with 200 µg human Cot-1 DNA (Invitrogen, Carlsbad, CA) and dried using a vacufuge concentrator (Eppendorf, Westbury, NY).
Channel 2
Source name
Genomic DNA isolated from cerebellum of subject 1 labeled with Cy3-dCTP.
Gender: male Age: 42 Tissue: brain-cerebellum Cause of death: acute cardiovascular failure
Extracted molecule
genomic DNA
Extraction protocol
genomic DNA isolation with phenol extraction according to standard protocol (Sambrook, J., Fritsch, E. F. & Maniatis, T. Molecular cloning; a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989)
Label
Cy3
Label protocol
Labeling was done using 2 mcg DNA, according to the BioPrime® Array CGH Genomic Labeling System protocol supplied by the manufacturer (Invitrogen, Carlsbad, CA). We used CY3 dCTP and CY5 dCTP respectively (PA53021 and PA55021, GE Healthcare, Piscataway, NJ). The labeled DNA probes were mixed together and combined with 200 µg human Cot-1 DNA (Invitrogen, Carlsbad, CA) and dried using a vacufuge concentrator (Eppendorf, Westbury, NY).
Hybridization protocol
The 32K slides were prehybridized in 5x SCC, 0.1% SDS at 45 ºC for at least one hour, washed with ddH20 and dried using compressed dust-free air. The dried pellets from the labeling steps were re-suspended in 50 µl of hybridization solution (50% Formamide, 4% SDS, 10 %, Dextran Sulfate, M.W. 500 000, 2 X SSC) and denatured for 5 min at 95°C, followed by incubation at 45°C for 2 h to block repetitive sequences. Subsequently, the probe mixture was applied to the slide surface, covered with a lifterslip (22x60mm, Erie Scientific, Portsmouth, NH) and hybridized at 45 °C for 20 hours in a slide chamber (Corning Inc. Life Sciences, Big Flats, NY). After hybridization the lifterslip was rinsed off using 1xPBS. Washing was performed in 25 % formamide, 2 X SSC, 0.1% SDS, at 45ºC, for 15 min, followed by 10 min in 1 X PBS at room temperature, and brief rinse in 0.2 X SSC. The array was immersed in deionized H2O and immediately dried using pressurized dust-free air.
Scan protocol
Image acquisition was performed using the GenePix 4000B scanner (Axon Instruments Inc, Union City, CA), and image analysis was carried out using the GenePixPro v6 software (Axon Instruments).
Description
none
Data processing
We uploaded the GPR files from GenePix pro into a pearl script called colloactor. This script links the information about clones to the array position. It also uploads the raw data to the BASE platform. These raw data files are attached as tables. Filtering and normalization was done using the LCB Data-WareHouse (dw.lcb.uu.se). The filters applied on the raw data removed spots containing more than 5% oversaturated pixels, spots with low Signal-to-Noise Ratio (SNR<3) and spots automatically and manually flagged as bad, absent or not found in GenePixPro program. Normalization was done using a print-tip loess method.
