|
| Status |
Public on Jul 01, 2011 |
| Title |
HEK293T_miR-neg_FSK1hr_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
HEK 293T Human embryonic kidney cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
transfected rna: negative control
agent: FSK
time: 1hr
|
| Treatment protocol |
Cells were reverse transfected with RNA using Lipofectamine2000 at a final concentration of 50 nM. Approximately 72hrs post-transfection, cells were treated with 15uM forskolin for 0, 1 or 4 hrs.
|
| Growth protocol |
HEK293T cells were grown in DMEM supplemented with 10% FBS and 1X PSG.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA (including small RNAs) was extracted using Trizol (Invitrogen) and the aqueous phase was purified with RNeasy Mini kit (Qiagen) according to the manufacturers’ protocols.
|
| Label |
Biotin
|
| Label protocol |
Second-strand cDNA synthesis was followed by in vitro transcription for linear amplification of each transcript and incorporation of biotinylated CTP and UTP.
|
| |
|
| Hybridization protocol |
The cRNA products were fragmented to 200 nucleotides or less, heated at 99 degrees C for 5 min and hybridized for 16 h at 45 degrees C. The microarrays were then washed at low (6X SSPE) and high (100mM MES, 0.1M NaCl) stringency and stained with streptavidin-phycoerythrin. Fluorescence was amplified by adding biotinylated anti-streptavidin and an additional aliquot of streptavidin-phycoerythrin stain.
|
| Scan protocol |
A confocal scanner was used to collect fluorescence signal at 3um resolution after excitation at 570 nm. The average signal from two sequential scans was calculated for each microarray feature.
|
| Data processing |
RMA normalized expression values calculated with Affymetrix Expression console
|
| |
|
| Submission date |
Oct 19, 2010 |
| Last update date |
Jul 01, 2011 |
| Contact name |
Anthony O Olarerin-George |
| E-mail(s) |
[email protected]
|
| Phone |
215-573-6197
|
| Fax |
215-573-9004
|
| Organization name |
University of Pennsylvania
|
| Department |
Pharmacology
|
| Lab |
Hogenesch Lab
|
| Street address |
835 BRB II/III, 421 Curie Blvd
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19153 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE24812 |
Impact of miR-132 and miR-381 on endogenous forskolin responsive CREB targets in HEK293T cells |
|
