|
| Status |
Public on Apr 29, 2006 |
| Title |
E19_epithelial cells_strain1 |
| Sample type |
RNA |
| |
|
| Source name |
E19 type II epithelial cells, 16 h, strain
|
| Organism |
Rattus norvegicus |
| Characteristics |
Strain: CD
Gender: male and female
Age: Embryonic day 19
Tissue: Fetal lung from time-pregnant rats
Cells: Type II epithelial
|
| Biomaterial provider |
Juan Sanchez-Esteban
|
| Treatment protocol |
After isolation, E19 type II cells were cultured on silastic membranes coated with collagen 1. Next day monolayers were exposed to cyclic strain 5%, 60 cycles/min for 15 min + 2.5% continuous distention for the remaining 45 min of each hour, for 16 h, using the Flexercell FX-4000 Strain Unit
|
| Growth protocol |
Cells were maintained in serum-free DMEM medium.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from E19 type II cells using TRIzol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA) and purified further using the Rneasy Mini Kit (Invitrogen).
|
| Label |
biotin
|
| Label protocol |
conversion of double stranded cDNA into biotin-labeled cRNA was accomplished using the BioArray High Yield RNA Transcript Labeling kit (T7) (Enzo Diagnostics, Farmingdale, NY) according to the manufacturer’s instructions.
|
| |
|
| Hybridization protocol |
After hybridization cocktails preparation, the Affymetrix rat E230A GeneChip array was hybridized with the fragmented labelled cRNA for 16 h at 45 °C as described in the Affymetrix Technical Analysis Manual.
|
| Scan protocol |
GeneChips, bound to streptavidin-phycoerythrin, were processed using the Affymetrix GeneChip Fluidics Station 400 and scanned with an Agilent G2500A GeneArray Scanner
|
| Description |
Total RNA was extracted from E19 type II cells using TRIzol reagent and the Rneasy Mini Kit. RNA was reverse-transcribed into cDNA with a T7-(dT)24 oligomer to prime the first-strand synthesis. DNA polymerase and DNA ligase were included in the synthesis of the second strand. After phase lock gel phenol/chloroform cDNA extraction and ethanol precipitation, double stranded cDNA was converted into cRNA. Following cleanup, fragmentation of the cRNA for target preparation was done according to the Affymetrix GeneChip Expression Analysis Protocol. After hybridization cocktails preparation, the Affymetrix rat E230A GeneChip array was hybridized with the fragmented labelled cRNA for 16 h at 45 °C as described in the Affymetrix Technical Analysis Manual. GeneChips, bound to streptavidin-phycoerythrin, were processed using the Affymetrix GeneChip Fluidics Station 400 and scanned with an Agilent G2500A GeneArray Scanner
|
| Data processing |
data were processed using Microarray Suite software, v. 5.0 (MAS 5.0) to yield expression values. These values were calculated from the difference in signal intensity following hybridization with with "perfect match" (PM) and "mismatch" (MM) oligonucleotides in each probe
|
| |
|
| Submission date |
Oct 31, 2005 |
| Last update date |
Nov 01, 2005 |
| Contact name |
Juan Sanchez-Esteban |
| E-mail(s) |
jsanchezesteban@wihri.org
|
| Phone |
401-2741122 ext1383
|
| Fax |
401-4537571
|
| Organization name |
Women & Infants Hospital/Brown Medical School
|
| Department |
Pediatrics
|
| Street address |
101 Dudley Street
|
| City |
Providence |
| State/province |
RI |
| ZIP/Postal code |
02905 |
| Country |
USA |
| |
|
| Platform ID |
GPL341 |
| Series (1) |
| GSE3541 |
DNA microarray reveals novel genes induced by mechanical forces in fetal lung type II epithelial cells |
|
