|
| Status |
Public on Feb 12, 2009 |
| Title |
Sample_22 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Adipose
|
| Organism |
Homo sapiens |
| Characteristics |
Adipose_Sample_22
Patient#7
Placebo_Fed
US-1013708
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the adipose samples by Qiagen RNeasy spin columns with DNAse treatment. 2 µg of each sample was precipitated in ethanol and re-suspended in 14 µl of water. Globin mitigation performed using Ambion's GLOBINclear kit (cat# AM1980; Austin, TX) according to manufacturer’s protocol. Briefly, biotinylated globin-specific oligonucleotides are mixed with total RNA and annealed to α and β globin transcripts; streptavidin-coated paramagnetic beads are then added to bind and capture the bound globin mRNAs; the beads are removed using a magnet. GLOBINclear-treated total RNA samples were adjusted to 150 µl, aliquots of which were used for qRT-PCR and microarray experiments.
|
| Label |
Cy3,Cy5
|
| Label protocol |
Custom automated version of the aminoallyl MessageAmp II kit from Ambion
|
| |
|
| Channel 2 |
| Source name |
Adipose
|
| Organism |
Homo sapiens |
| Characteristics |
US-1021605
common reference
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from the adipose samples by Qiagen RNeasy spin columns with DNAse treatment. 2 µg of each sample was precipitated in ethanol and re-suspended in 14 µl of water. Globin mitigation performed using Ambion's GLOBINclear kit (cat# AM1980; Austin, TX) according to manufacturer’s protocol. Briefly, biotinylated globin-specific oligonucleotides are mixed with total RNA and annealed to α and β globin transcripts; streptavidin-coated paramagnetic beads are then added to bind and capture the bound globin mRNAs; the beads are removed using a magnet. GLOBINclear-treated total RNA samples were adjusted to 150 µl, aliquots of which were used for qRT-PCR and microarray experiments.
|
| Label |
Cy3,Cy5
|
| Label protocol |
Custom automated version of the aminoallyl MessageAmp II kit from Ambion
|
| |
|
| |
| Hybridization protocol |
Microarrays are incubated at 40°C for 48 hours in a rotating carousel. Hybridizations to custom Agilent microarrays are completed as previously described (Hughes et al.Nat Biotech (2001), 19(4):342-7). Microarrays are washed to remove non-specific hybridized sample. Afterwards, microarrays are dried in an ozone-free nitrogen chamber.
|
| Scan protocol |
Microarrays are scanned using the Agilent LP2 laser scanner. The scanner output is a Tiff file, which contains the quantitative hybridization data from each individual microarray. The Tiff files are then processed using Rosetta custom feature extraction software.
|
| Description |
VALUEs represent averaged data for both channels from a fluor-reverse pair
|
| Data processing |
Data were processed using the Rosetta Resolver® system. Rosetta's custom feature extraction software performs error modeling before data are loaded into the Resolver system. The Resolver system performs a squeeze operation that combines replicates of the same sequence in an array while applying error weighting. The error weighting consists of adjusting for additive and multiplicative noise. A P-value is generated and propagated throughout the system. The P-value represents the probability that a gene is expressed. The Resolver system allows users to set thresholds, below which genes of a P-value are considered to be significantly expressed. The Resolver system also combines multiple arrays using a squeezing process. If multiple spots reference one sequence, summarization is performed using an error-weighted average as described in Roland Stoughton and Hongyue Dai, Statistical Combining of Cell Expression Profiles. US Patent #6,351,712, February 26, 2002.
|
| |
|
| Submission date |
Feb 15, 2008 |
| Last update date |
Feb 15, 2008 |
| Contact name |
Michael Nebozhyn |
| Organization name |
Merck, Inc
|
| Department |
GpGx/Computational System biology
|
| Street address |
Mail Stop WP53B-120, 770 Sumneytown Pike, Building 53, P.O. Box 4
|
| City |
West Point |
| State/province |
PA |
| ZIP/Postal code |
19486 |
| Country |
USA |
| |
|
| Platform ID |
GPL3991 |
| Series (1) |
| GSE10545 |
Profiling of Human Adipose Tissue and Effect of Sibutramine on Gene Expression |
|
