|
| Status |
Public on Dec 25, 2011 |
| Title |
ADSV_28_PPARG_3 |
| Sample type |
RNA |
| |
|
| Source name |
ADSV_28_derived_MPCs_+PPAR2_rep3
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: MPC
agent: PPAR2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA from human cell lines or human fat was extracted with Trizol (Invitrogen) and purified via the RNeasy mini kit (Qiagen) according to the manufacturers’ instructions. RNA quality was assessed using an Aglient Bioanalyzer
|
| Label |
cy5
|
| Label protocol |
Cyanine-3\Cyanine-5 labeled cRNA was prepared from 0.3 ug total RNA using the Two-Color Low-Input QuickAmp Labeling kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ng of cRNA (specific activity >10.0 pmol Cy3/5/ug cRNA) per channel per array spot (8 per slide) (cy3 and cy5 channels both utilized) was fragmented at 60°C for 30 minutes following the manufacturers instructions. On completion of the fragmentation reaction, 1 volume of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Sureprint G3 8x60k gene expression arrays (G4858A-028004) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent)(both without Triton added).
|
| Scan protocol |
Slides were scanned immediately after washing and placement within an Agilent Ozone Barrier Slide cover (Agilent) the Agilent DNA Microarray Scanner (G2505C) using two color scan setting for 8x60k array Scan resolution 3 microns. Scanning was done in both channels and PMT is set to 100% for both Cy3\cy5 channels.
|
| Description |
Gene Expression
Raw data file:
252800411053_201102140806_S01_GE2_105_Dec08_1_2.txt
Data column in raw data file: rProcessedSignal (AA)
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE2-105_Dec08 with the cognate G4858A-028004 grid file to obtain background subtracted and spatially detrended Processed Signal intensities. Processed Signal Intensities were expressed as a ratio to the 75th percentile value (control probes excluded) and median centered for each array and log2 transformed. Only probes tagged by Feature Extraction as above background in all constituents of a sample triplicate or duplicate set in at least one condition were considered for further analysis. After background filtering, probes values were collapsed by the GeneName column from the Feature Extraction output to condense multiple reads of the same gene. To correct for slide batch effects (each slide = 8 arrays) the R ComBat package (Adjusting batch effects in microarray expression data using empirical Bayes methods. Biostatistics 2007) was employed using the universal reference (technical replicates) array on each slide upon which a universal replicate was hybridized along with each condition treating each array as a different batch.
|
| |
|
| Submission date |
Jun 16, 2011 |
| Last update date |
Dec 25, 2011 |
| Contact name |
Tim D Ahfeldt |
| E-mail(s) |
tim.ahfeldt@mssm.edu
|
| Organization name |
Icahn School of Medicine at Mount Sinai
|
| Department |
Neuroscience
|
| Lab |
Tim D Ahfeldt
|
| Street address |
1425 Madison Ave
|
| City |
NYC |
| State/province |
NY |
| ZIP/Postal code |
10029 |
| Country |
USA |
| |
|
| Platform ID |
GPL13607 |
| Series (2) |
| GSE30039 |
Programming human pluripotent stem cells into adipocytes [Agilent] |
| GSE30041 |
Programming human pluripotent stem cells into adipocytes |
|
