|
| Status |
Public on Jul 29, 2010 |
| Title |
aERKO V vs HPTE 2hr |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
aERKO uterus sesame oil V
|
| Organism |
Mus musculus |
| Characteristics |
esr1 genotype: aERKO (Esr1 null)
strain: C57Bl/6J
age: adult (10+ weeks)
gender: ovariectomized female
|
| Treatment protocol |
ovariectomized mice were allowed to rest for 10-14 days, then injected with saline or 250ng estradiol, 100 ul sesame oil or 750 ug BPA or HPTE in 100 ul oil ip. Uterine tissue was collected 2 or 24 hours after injection and frozen in liquid nitrogen
|
| Extracted molecule |
total RNA |
| Extraction protocol |
pools of 3 frozen uteri were pulverized and homogenized in Trizol (Invitrogen 5- 100 mg tissue/ml Trizol, minimum 800 ul) . Extraction proceeded as descibed in manufacturer's protocol. Following resuspension of RNA pellet, a second preciptation was done with NaAcetate and ethanol. This pellet was then resuspended in depc treated water and cleaned up using the Qiagen RNeasy kit clean up protocol. RNA concentration and A260/280 was checked and quality was evaluated by formaldehyde gel.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy3 labeled cRNA and Cy5 labeled cRNA were produced according to manufacturer’s protocol.
|
| |
|
| Channel 2 |
| Source name |
aERKO uterus sesame oil HPTE 2h
|
| Organism |
Mus musculus |
| Characteristics |
esr1 genotype: aERKO (Esr1 null)
strain: C57Bl/6J
age: adult (10+ weeks)
gender: ovariectomized female
|
| Treatment protocol |
ovariectomized mice were allowed to rest for 10-14 days, then injected with saline or 250ng estradiol, 100 ul sesame oil or 750 ug BPA or HPTE in 100 ul oil ip. Uterine tissue was collected 2 or 24 hours after injection and frozen in liquid nitrogen
|
| Extracted molecule |
total RNA |
| Extraction protocol |
pools of 3 frozen uteri were pulverized and homogenized in Trizol (Invitrogen 5- 100 mg tissue/ml Trizol, minimum 800 ul) . Extraction proceeded as descibed in manufacturer's protocol. Following resuspension of RNA pellet, a second preciptation was done with NaAcetate and ethanol. This pellet was then resuspended in depc treated water and cleaned up using the Qiagen RNeasy kit clean up protocol. RNA concentration and A260/280 was checked and quality was evaluated by formaldehyde gel.
|
| Label |
Cy5
|
| Label protocol |
Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy3 labeled cRNA and Cy5 labeled cRNA were produced according to manufacturer’s protocol.
|
| |
|
| |
| Hybridization 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.
|
| Scan protocol |
Slides were washed as indicated in the Agilent 60-mer oligo microarray processing protocol and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software, using defaults for all parameters.
|
| Description |
US22502532_251486813435_S02_H.tif
US22502532_251486813435__S02_L.tif
gene expression in aERKO ovariectomized oil vehicle vs HPTE 2h treated uterus
|
| Data processing |
Data was obtained using the Agilent Feature Extraction software (v9.1), using defaults for all parameters. The default setting for the protocol we used is polynomial data fit.
|
| |
|
| Submission date |
Jul 27, 2010 |
| Last update date |
Jul 29, 2010 |
| Contact name |
NIEHS Microarray Core |
| E-mail(s) |
microarray@niehs.nih.gov, liuliw@niehs.nih.gov
|
| Organization name |
NIEHS
|
| Department |
DIR
|
| Lab |
Microarray Core
|
| Street address |
111 T.W. Alexander Drive
|
| City |
RTP |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE18168 |
Uterine gene profiles from WT, KIKO (DNA-binding deficient ERα) and aERKO mice treated with estradiol or xenoestrogens |
|
