|
| Status |
Public on Jun 25, 2009 |
| Title |
Cu_400uM_4h_Rep 5 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Control 4h
|
| Organism |
Homo sapiens |
| Characteristics |
Control
|
| Treatment protocol |
HepG2 cells were plated and allowed to grow until they were 50 % confluent. Cells were then treated with 100, 200, 400 and 600 mM of copper sulfate for 4, 8, 12 and 24 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were isolated from untreated and treated cells using RNeasy mini kits (Qiagen, Inc.) following manufacturer's instructions. The quality of the purified RNA was determined using a BioAnalyzer (Agilent Technologies, Palo Alto, CA).
|
| Label |
CY3
|
| Label protocol |
Total RNA (100 ng) from control or metal-treated cells was amplified and labeled with Cy3 or Cy5 fluorescent dye using Agilent Technologies Low RNA Input Linear Amplification labeling kit following the manufacture's protocol.
|
| |
|
| Channel 2 |
| Source name |
Cu_400uM_4h
|
| Organism |
Homo sapiens |
| Characteristics |
Treated
|
| Treatment protocol |
HepG2 cells were plated and allowed to grow until they were 50 % confluent. Cells were then treated with 100, 200, 400 and 600 mM of copper sulfate for 4, 8, 12 and 24 h.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNAs were isolated from untreated and treated cells using RNeasy mini kits (Qiagen, Inc.) following manufacturer's instructions. The quality of the purified RNA was determined using a BioAnalyzer (Agilent Technologies, Palo Alto, CA).
|
| Label |
CY5
|
| Label protocol |
Total RNA (100 ng) from control or metal-treated cells was amplified and labeled with Cy3 or Cy5 fluorescent dye using Agilent Technologies Low RNA Input Linear Amplification labeling kit following the manufacture's protocol.
|
| |
|
| |
| Hybridization protocol |
The quantity and purity of the resulting fluorescently-labeled cRNA was evaluated using a Nanodrop ND-100 spectrophotometer (Nanodrop Technologies), and the size distribution using an Agilent Bioanalyzer. Equal amounts of Cy3- and Cy5-lableled cRNA were then hybridized to an Agilent Human Microarray (~22,000 k features) for 17 h at 65°C.
|
| Scan protocol |
The hybridized microarrays were washed and scanned using an Agilent G2565BA scanner. Data were extracted from the scanned images using Agilent Feature Extraction software.
|
| Description |
Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 100ng of total RNA, Cy3 or Cy5 labeled cRNA was produced according to manufacturer's protocol. For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Chips were scanned with an Agilent Scanner and processed with the Agilent Feature Extraction Software.
|
| Data processing |
GeneSpring GX 7.3 (Agilent Technologies) was used to identify genes that showed significant changes in gene expression with any treatment. For global normalization of raw microarray data, per spot and per chip: intensity dependent (Lowess) normalization was applied.
|
| |
|
| Submission date |
Nov 06, 2007 |
| Last update date |
Aug 14, 2011 |
| Contact name |
Min Ok Song |
| E-mail(s) |
songm2@niehs.nih.gov
|
| Phone |
919-541-2679
|
| Fax |
919-541-5737
|
| Organization name |
NIEHS
|
| Department |
DIR ETP
|
| Lab |
LTEG
|
| Street address |
111 T.W. Alexander Dr
|
| City |
RTP |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL6087 |
| Series (1) |
| GSE9539 |
Global transcriptome changes in HepG2 cells exposed to copper |
|
