|
| Status |
Public on Jun 15, 2009 |
| Title |
Wildtype_30m_Heatshock_rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
CHIP(+/+) MEF, heat shock, 30 min
|
| Organism |
Mus musculus |
| Characteristics |
embryonic fibroblast
|
| Treatment protocol |
MEF cultures were either maintained at 37° C or heat shocked at 43° C for the indicated time points prior to RNA harvesting.
|
| Growth protocol |
MEFs (mouse embryonic fibroblasts) isolated from day 13.5 embryos of C57BL6 CHIP (+/-) crosses were grown under similar conditions with the addition of β-mercaptoethanol at 37° C, 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified using the RNeasy microkit (Qiagen) in accordance with the manufacturer’s instructions which included DNase treatment to eliminate genomic contamination. RNA quantity, purity and integrity were assessed by spectrophotometry and microcapillary electrophoresis on an Agilent BioAnalyzer.
|
| Label |
Cy5
|
| Label protocol |
500 ng of total RNA from three biological replicates from each condition were used for amplification and labeled with Cy5 using the Agilent Low Input RNA amplification kit exactly as described by the manufacturer.
|
| |
|
| Channel 2 |
| Source name |
Stratagene UMRR
|
| Organism |
Mus musculus |
| Characteristics |
pool of mouse reference RNA
|
| Treatment protocol |
MEF cultures were either maintained at 37° C or heat shocked at 43° C for the indicated time points prior to RNA harvesting.
|
| Growth protocol |
MEFs (mouse embryonic fibroblasts) isolated from day 13.5 embryos of C57BL6 CHIP (+/-) crosses were grown under similar conditions with the addition of β-mercaptoethanol at 37° C, 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified using the RNeasy microkit (Qiagen) in accordance with the manufacturer’s instructions which included DNase treatment to eliminate genomic contamination. RNA quantity, purity and integrity were assessed by spectrophotometry and microcapillary electrophoresis on an Agilent BioAnalyzer.
|
| Label |
Cy3
|
| Label protocol |
500 ng of total RNA from three biological replicates from each condition were used for amplification and labeled with Cy5 using the Agilent Low Input RNA amplification kit exactly as described by the manufacturer.
|
| |
|
| |
| Hybridization protocol |
Labeled cRNA was co-hybridized to Agilent G4122F Whole Mouse Genome 44K oligonucleotide arrays with equimolar amounts of Cyanine-3 labeled UMRR. Oligoarray control targets and hybridization buffer (Agilent In Situ Hybridization Kit Plus) were added, and samples were applied to microarrays enclosed in Agilent SureHyb-enabled hybridization chambers. After hybridization, slides were washed sequentially.
|
| Scan protocol |
Scanned on an Axon Instruments GenePix 4000b scanner.
|
| Description |
none
|
| Data processing |
The data were processed using Feature Extraction software and normalized using per-chip and per-spot intensity-dependent LOWESS normalization with a threshold of 0.01. The data at the two heat shock time-points, 30 and 240 minutes, were normalized by their respective un-shocked controls.
|
| |
|
| Submission date |
Jan 08, 2009 |
| Last update date |
Jun 15, 2009 |
| Contact name |
Jonathan C Schisler |
| E-mail(s) |
schisler@unc.edu
|
| Phone |
919-843-8708
|
| Organization name |
The University of North Carolina at Chapel Hill
|
| Department |
McAllister Heart Institute
|
| Lab |
Schisler Lab
|
| Street address |
MBRB, Rm 2340C
|
| City |
Chapel Hill |
| State/province |
NC |
| ZIP/Postal code |
27599-7126 |
| Country |
USA |
| |
|
| Platform ID |
GPL7202 |
| Series (1) |
| GSE14339 |
Stress-dependent CHIP/Daxx interaction suppresses the p53 apoptotic program |
|
