|
| Status |
Public on Sep 15, 2009 |
| Title |
P43_TGFB_T03 (baseline, SSc FBs) |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
P43_TGFB_T01 (baseline)
|
| Organism |
Homo sapiens |
| Characteristics |
primary fibroblasts from 41yo male, 1 year diagnosed with dSSc, forearm biopsy taken in 2000
|
| Biomaterial provider |
Kari Connolly UCSF
|
| Treatment protocol |
Baseline measurement (3 of 3 triplicates in 24 hour TGFB timecourse)
|
| Growth protocol |
Plated at 4x10e4 - grown for 48 hours in DMEM + Pen/Strep + 10% FBS (37C 5% CO2)
Grown for 24 hours in low serum conditions (DMEM + Pen/Strep + 0.1%FBS, 37C 5%CO2)
- then either treated with 50pM TGFB or sample taken for baseline measurement.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNeasy mini columns with on column DNaseI digestion (Qiagen)
|
| Label |
Cy3
|
| Label protocol |
Agilent Low RNA Input Linear Amplification protocol: 200 ng of total RNA from each time point sample was converted to Cy3-CTP (Perkin Elmer) labeled cRNA, and Universal Human Reference (UHR) RNA (Stratagene) was converted to Cy5-CTP (Perkin Elmer) using a low input linear amplification kit (Agilent Technologies) as per manufacturers protocols. Labeled cRNA targets were then purified using RNeasy columns (Qiagen).
|
| |
|
| Channel 2 |
| Source name |
Universal Human Reference RNA
|
| Organism |
Homo sapiens |
| Characteristics |
pool of RNA from 10 different human cell lines, commercially available from Stratagene.
|
| Biomaterial provider |
purchased from Stratagene
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Stratagene's protocol
|
| Label |
Cy5
|
| Label protocol |
Agilent Low RNA Input Linear Amplification protocol: 200 ng of total RNA from each time point sample was converted to Cy3-CTP (Perkin Elmer) labeled cRNA, and Universal Human Reference (UHR) RNA (Stratagene) was converted to Cy5-CTP (Perkin Elmer) using a low input linear amplification kit (Agilent Technologies) as per manufacturers protocols. Labeled cRNA targets were then purified using RNeasy columns (Qiagen).
|
| |
|
| |
| Hybridization protocol |
Agilent Gene Expression Hybridization kit was used for hybridization. Cy3- and Cy5-labeled cRNA was hybridized to stripped 4 x 44K Agilent Whole Human genome oligo-array. Hybridization was carried out for 17 hours at 65oC with rotation.
|
| Scan protocol |
Microarrays were scanned using a dual laser GenePix 4000B scanner (Axon Instruments). The pixel intensities of the acquired images were then quantified using GenePix Pro 5.1 software.
|
| Description |
Comparison of dermal fibroblasts from healthy and dSSc patients treated and untreated with TGFB.
|
| Data processing |
Arrays were visually inspected for defects or technical artifacts, and poor quality spots were manually flagged and excluded from further analysis. Only spots with fluorescent signal at least 1.5 fold greater than local background in both Cy3- Cy5- channels were included in the analysis. Genes missing more than 20% of their data points were excluded, resulting in 18,695 probes that passed the filtering criteria.
|
| |
|
| Submission date |
Aug 18, 2008 |
| Last update date |
Aug 03, 2009 |
| Contact name |
Jennifer L Sargent |
| E-mail(s) |
jennifer.sargent@gmail.com
|
| Organization name |
Dartmouth Medical School
|
| Department |
Genetics
|
| Lab |
Whitfield
|
| Street address |
Dartmouth College; HB7400
|
| City |
Hanover |
| State/province |
NH |
| ZIP/Postal code |
03755 |
| Country |
USA |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE12493 |
A TGFB Responsive Gene Expression Signature is Associated With More Severe Skin and Lung Disease in Diffuse Scleroderma |
|
