|
| Status |
Public on Aug 01, 2011 |
| Title |
Human blood_Acute ITP_Pt5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
ITP patient peripheral blood (test)
|
| Organism |
Homo sapiens |
| Characteristics |
patient: 5
tissue: Whole blood
disease: ITP
disease state: Self-limited acute ITP
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using PAXgene Blood RNA Kit following manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
The total RNA from ITP paitents and Universal Human Reference RNA was first linerly amplified for one round using MessageAmp aRNA Amplification Kit(Ambion) following manufacturer's protocol, then 6µg of amplified aRNA was mixed with 10µg of pdN6 hexamer primer, incubate at 65°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dCTP, dGTP, with 200 µM dTTP, 100 µM Cy3 or Cy5-labeled (Amersham) dUTP.
|
| |
|
| Channel 2 |
| Source name |
Universal Human Reference RNA, Stratagene
|
| Organism |
Homo sapiens |
| Characteristics |
reference: Total RNA from 10 pooled cell lines
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using PAXgene Blood RNA Kit following manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
The total RNA from ITP paitents and Universal Human Reference RNA was first linerly amplified for one round using MessageAmp aRNA Amplification Kit(Ambion) following manufacturer's protocol, then 6µg of amplified aRNA was mixed with 10µg of pdN6 hexamer primer, incubate at 65°C for 10 min, then reversed transcribed at 42°C for 1 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dCTP, dGTP, with 200 µM dTTP, 100 µM Cy3 or Cy5-labeled (Amersham) dUTP.
|
| |
|
| |
| Hybridization protocol |
For each experimental sample, Cy5- and Cy3-labeled samples were co-hybridized to a Stanford cDNA microarray at 65°C for 14 hours. After hybridization, arrays were washed before scanning.
|
| Scan protocol |
Microarrays were imaged using an Axon GenePix 4000 scanner (Axon Instruments) and fluorescence ratios for array elements were extracted using GenePix software.
GPR file: ITP 5_SHGO.gpr (see below for link to file)
|
| Description |
Peripheral blood from ITP Pt#5. Collected within 6 months of dx during active disease, the pt recovered within 6 months of dx.
|
| Data processing |
Normalization performed with GenePix Pro software version is 5.0. The fluorescence ratios were normalized by mean-centering genes for each array (that is, 'global' normalization), and then by mean centering each gene across all arrays.
The reason for the discrepancy between the array description and the platform data table is mainly due to the filters we set up for data retrieval from SMD: Select only features with no flag (include only features that have not been designated as unreliable either by the scanning software or by the array/hybridization owner); Regression correlation>0.6;median fluorescent hybridization signal intensity divided by median background intensity >1.5 in both the sample and reference channels for at least 80% of the samples analyzed. The controls account for a relatively small portion of the missing data.
|
| |
|
| Submission date |
Aug 23, 2010 |
| Last update date |
Aug 01, 2011 |
| Contact name |
Bing Zhang |
| Organization name |
Stanford University
|
| Street address |
3373 Hillview Ave
|
| City |
Palo Alto |
| State/province |
CA |
| ZIP/Postal code |
94304 |
| Country |
USA |
| |
|
| Platform ID |
GPL10845 |
| Series (1) |
| GSE23754 |
Acute ITP vs. Chronic ITP (Children) |
|
