|
| Status |
Public on Apr 01, 2008 |
| Title |
SV12-72, SV40EA transfected |
| Sample type |
RNA |
| |
|
| Source name |
human fetal lung fibroblast TIG-1 clone SV12 transfected with SV40EA
|
| Organism |
Homo sapiens |
| Characteristics |
human fetal lung fibroblast TIG-1 clone SV12 at 72 PDL transfected with SV40EA
|
| Biomaterial provider |
Health Science Research Resources Bank, Tokyo, Japan
|
| Treatment protocol |
SV40 early antigens (SV40EA) expression vector was transfected to TIG-1 and isolated as a G418 resistant clone. Exponentially growing cultured cells were collected using trypsin followed by rinse with PBS, and freezed until use.
|
| Growth protocol |
Cells were cultured in Dulbecco's modified Eagle's minimal essential medium (DMEM; Sigma-Aldrich Japan, Tokyo, Japan) containing 10% heat-inactivated fetal bovine serum (FBS; BioWhittaker, Verviers, Belgium) in a humidified atmosphere of 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from the frozen cultured cell pellets using QIAGEN RNeasy mini kit (QIAGEN, Inc., Valencia, CA), respectively, according to the manufacturer's protocols. The quality of the RNA was checked using Agilent Technologies 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
|
| Label |
biotin
|
| Label protocol |
The first-strand cDNA was generated from 1 microgram of total RNA using reverse transcriptase and a T7 primer using the CodeLink Expression Assay Reagent Kit, and then second-strand cDNA was produced using DNA polymerase mix and RNase H. cRNA (complementary RNA) was generated via an in vitro transcription reaction using T7 RNA polymerase and biotin-11-UTP.
|
| |
|
| Hybridization protocol |
The synthesized 10 microgram of cRNA, quantified by spectrometry and qualified using an Agilent 2100 Bioanalyzer, was then fragmented and hybridized to each array. After 16hr hybridization, arrays were rinsed with 0.75 x TNT buffer for 1 h, labeled with Streptavidin-Cy5 at room temperature for 30 min, rinsed again with TNT buffer and 0.05% Tween 20, and dried by centrifugation.
|
| Scan protocol |
Each microarray was scanned using using an Agilent DNA Microarray Scanner.
|
| Description |
mortal lifespan, transformed
|
| Data processing |
Expression levels were quantified by CodeLink Expression Analysis Software (GE Healthcare) and normalized to the median expression value of the whole array spots using GeneSpringTM GX (Agilent Technologies).
|
| |
|
| Submission date |
Sep 17, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Keiko Hiyama |
| E-mail(s) |
khiyama@hiroshima-u.ac.jp
|
| Phone |
81-82-257-5841
|
| Fax |
81-82-256-7105
|
| Organization name |
Hiroshima University
|
| Department |
Research Institute for Radiation Biology and Medicine
|
| Lab |
Dept. Translational Cancer Research
|
| Street address |
1-2-3 Kasumi, Minami-ku
|
| City |
Hiroshima |
| State/province |
Hiroshima |
| ZIP/Postal code |
734-8553 |
| Country |
Japan |
| |
|
| Platform ID |
GPL1928 |
| Series (1) |
| GSE9077 |
Expression profiles of immortal lung fibroblasts |
|
