|
| Status |
Public on Apr 07, 2011 |
| Title |
T/T+MAA |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
RNA prepared using Trizol from mouse TM3 cells infected by retrovirus encoding Androgen Receptor (AR) cDNA.
|
| Organism |
Mus musculus |
| Characteristics |
cell line: TM3 Leydig cell infected by retrovirus encoding Androgen Recetor (AR) cDNA.
treatment: 24h-testosterone-10 nM
|
| Treatment protocol |
TM3-AR cells were treated for 24 hr with either testosterone (T, 10 nM) or MAA (5 mM), or with testosterone in combination with MAA (T+MAA), or were untreated (UT).
|
| Growth protocol |
TM3 and TM3-AR cells (see below) were grown in DMEM-F12 medium containing 5% horse serum and 2.5% FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of RNA
|
| Label |
Alexa 647
|
| Label protocol |
RNA samples were amplified as antisense-RNA (aRNA) while incorporating aminoallyl modified bases using the TargetAMP 1-Round Aminoallyl-aRNA Amplification Kit 101 per the vendor’s protocol (Epicentre, Madison, WI). Five µg of each aminoallyl-aRNA sample was fluorescent labeled using Alexa 555 or Alexa 647 by incubating with an amine-reactive dye conjugate for 1 hr at room temperature. Unincorporated dye was removed using an RNeasy column (Qiagen, Valencia, CA). Dye incorporation efficiency was determined using a Nanodrop spectrophotometer.
|
| |
|
| Channel 2 |
| Source name |
TM3-AR Leydig cell RNA prepared using Trizol
|
| Organism |
Mus musculus |
| Characteristics |
cell line: TM3-AR Leydig cell
treatment: 24h-testosterone-10 nM and MAA-5mM
|
| Treatment protocol |
TM3-AR cells were treated for 24 hr with either testosterone (T, 10 nM) or MAA (5 mM), or with testosterone in combination with MAA (T+MAA), or were untreated (UT).
|
| Growth protocol |
TM3 and TM3-AR cells (see below) were grown in DMEM-F12 medium containing 5% horse serum and 2.5% FBS.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol extraction of RNA
|
| Label |
Alexa 555
|
| Label protocol |
RNA samples were amplified as antisense-RNA (aRNA) while incorporating aminoallyl modified bases using the TargetAMP 1-Round Aminoallyl-aRNA Amplification Kit 101 per the vendor’s protocol (Epicentre, Madison, WI). Five µg of each aminoallyl-aRNA sample was fluorescent labeled using Alexa 555 or Alexa 647 by incubating with an amine-reactive dye conjugate for 1 hr at room temperature. Unincorporated dye was removed using an RNeasy column (Qiagen, Valencia, CA). Dye incorporation efficiency was determined using a Nanodrop spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
Hybridization was performed using 0.825 µg of Alexa 555-labeled aRNA and 0.825 µg of Alexa 647-labeled aRNA. Agilent’s SureHyb hybridization chambers were used in a hybridization oven and rotation rack for 17 hr at 65° C at 10 rpm. After hybridization, the slides were washed per Agilent’s SSPE wash protocol.
|
| Scan protocol |
Slides were scanned using an Agilent dual laser scanner using the extended dynamic range option, which utilizes two 5 m scans of each slide at settings of PMT 100% and PMT 10% to increase signal dynamic range and avoid feature saturation. TIFF images were analyzed using Agilent’s feature extraction software (version 9.5.3, protocol GE2-v5_91_0806).
|
| Description |
RNA expression profile for testosterone treated vs. testosterone+MAA treated mouse TM3 cells infected by retrovirus containing AR cDNA.
|
| Data processing |
Linear and LOWESS normalization as determined using Agilent's Feature Extraction software
|
| |
|
| Submission date |
Feb 18, 2011 |
| Last update date |
Apr 07, 2011 |
| Contact name |
David J. Waxman |
| E-mail(s) |
djw@bu.edu
|
| Organization name |
Boston University
|
| Department |
Department of Biology and Bioinformatics Program
|
| Street address |
5 Cummington Mall
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE27410 |
Complex modulation of androgen responsive gene expression by methoxyacetic acid |
|
