|
| Status |
Public on May 21, 2008 |
| Title |
Astro Total Control_1 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
primary normal astrocytes; total RNA; untreated
|
| Organism |
Homo sapiens |
| Characteristics |
Primary normal human astrocytes as control
|
| Biomaterial provider |
Cambrex Bioscience
|
| Treatment protocol |
Treatment: no treatment, as a control grown in the same condition as treatment group
|
| Growth protocol |
Primary normal human astrocytes were purchased from Cambrex Bioscience and grown in astrocyte Growth Media for no more than three weeks with a change of media every 2 days until cells were 80% confluent. All cultures are maintained at 37 C° in an atmosphere of 5% CO2 and 95% room air.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cells were scraped from tissue culture flasks, and total RNA was extracted from each sample using TRIZOL reagent (Invitrogen, Carlsbad, CA) passed through an RNeasy spin column (Qiagen, Valencia, CA) and then amplified using RiboAmp RNA kits (Arcturus, Mountain View, CA) according to manufacturer’s protocol.
|
| Label |
CY3
|
| Label protocol |
Amplified RNA (1.5-3.0 Ag) was labeled with Cy3-dUTP (experimental RNA) or Cy5-dUTP (Stratagene Universal Reference, La Jolla, CA) using Superscript II Reverse Transcriptase (Invitrogen, Carlsbad, CA). Each cDNA microarray chip contained 7680 human cDNA clones (National Cancer Institute ROSP 8K Human Array), and methods for microarray hybridization and washing were described previously (Chuang YY, Chen Y, Gadisetti, et al. Gene expression after treatment with hydrogen peroxide, menadione, or t –butyl hydroperoxide in breast cancer cells. Cancer Res 2002;62:6246–54.16).
|
| |
|
| Channel 2 |
| Source name |
Human Reference RNA
|
| Organism |
Homo sapiens |
| Characteristics |
Stratagene’s Universal Human Reference RNA is composed of total RNA from 10 human cell lines. The reference RNA is designed to
be used as a reference for microarray gene-profiling experiments.
|
| Biomaterial provider |
Stratagene
|
| Treatment protocol |
no treatment
|
| Growth protocol |
reference to the manufacturer
|
| Extracted molecule |
total RNA |
| Extraction protocol |
PROTOCOL
Universal Human Reference RNA is provided in a solution of 70% ethanol and 0.1 M sodium acetate. Prepare the Reference RNA for
use as follows:
1. Centrifuge the tube at 12,000 × g for 15 minutes at 4°C.
2. Carefully remove the supernatant.
3. Wash the pellet in 70% ethanol.
4. Centrifuge the tube at 12,000 × g for 15 minutes at 4°C.
5. Carefully remove the supernatant and air-dry the pellet at room temperature for 30 minutes to remove retained ethanol.
6. Resuspend the pellet in RNase-free water to the desired concentration.
|
| Label |
CY5
|
| Label protocol |
Amplified RNA (1.5-3.0 Ag) was labeled with Cy3-dUTP (experimental RNA) or Cy5-dUTP (Stratagene Universal Reference, La Jolla, CA) using Superscript II Reverse Transcriptase (Invitrogen, Carlsbad, CA). Each cDNA microarray chip contained 7680 human cDNA clones (National Cancer Institute ROSP 8K Human Array), and methods for microarray hybridization and washing were described previously (Chuang YY, Chen Y, Gadisetti, et al. Gene expression after treatment with hydrogen peroxide, menadione, or t –butyl hydroperoxide in breast cancer cells. Cancer Res 2002;62:6246–54.16).
|
| |
|
| |
| Hybridization protocol |
methods for microarray hybridization and washing were described previously (Chuang YY, Chen Y, Gadisetti, et al. Gene expression after treatment with hydrogen peroxide, menadione, or t –butyl hydroperoxide in breast cancer cells. Cancer Res 2002;62:6246–54.16).
|
| Scan protocol |
Hybridized arrays were scanned with 10-Am resolution on a GenePix 4000A scanner (Axon Instruments, Inc., Foster City, CA) at wavelengths 635 and 532 nm for Cy5- and Cy3-labeled probes, respectively. The resulting TIFF images were analyzed by GenePix Pro 4.0 software (Axon Instruments).
|
| Description |
Tumor cell lines had a biological replicate, and each replicate was run on duplicate slides.
|
| Data processing |
The ratios of the sample intensity to the reference [green (Cy3)/red (Cy5)] intensity for all targets were determined, and ratio normalization was done to normalize the center of ratio distribution to 1.0.
|
| |
|
| Submission date |
May 12, 2008 |
| Last update date |
May 21, 2008 |
| Contact name |
Shuping Zhao |
| E-mail(s) |
zhaos@mail.nih.gov
|
| Phone |
301-594-5309
|
| Fax |
301-402-3134
|
| Organization name |
NCI
|
| Department |
Radiation Oncology Branch
|
| Street address |
8717 Grovemont Circle
|
| City |
Gaithersburg |
| State/province |
MD |
| ZIP/Postal code |
20877 |
| Country |
USA |
| |
|
| Platform ID |
GPL6665 |
| Series (1) |
| GSE11517 |
Radiation-Induced Changes in Gene Expression Involve Recruitment of Existing Messenger RNAs to and from Polysomes |
|
