|
| Status |
Public on Dec 31, 2010 |
| Title |
oAPSp1b - pool of anterior lobe prostate stroma laser capture microdissected from three old (20-24 month-old) mice |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
MGS
|
| Organism |
Mus musculus |
| Characteristics |
gender: male
strain: Swiss-Webster
age: Adult
tissue: reference pool of normal adult tissues (10% prostate and 30% each testis, liver, and kidney)
amplification: one round aRNA
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Reference RNA was purified using Trizol (Life Technologies, Rockville, MD) following the manufacturer’s protocol. The RNA was then further purified by RNeasy maxi kit (Qiagen Inc, Valencia, CA) and treated with DNAse using the RNase-Free DNase Set (Qiagen Inc, Valencia, CA) followed by one round of mRNA amplification with amino-allyl incorporation using the Ambion MessageAmp aRNA Kit (Ambion Inc, Austin, TX), according to the manufacturer’s specifications.
|
| Label |
Cy5
|
| Label protocol |
A total of 825 ng of amplified amino-allyl aRNA from each experimental sample was labeled with Cy3 fluorescent dye (reference amino-allyl aRNA was labeled with Cy5) following Agilent suggested protocols.
|
| |
|
| Channel 2 |
| Source name |
oAPSp1b
|
| Organism |
Mus musculus |
| Characteristics |
gender: male
strain: C57BL/6
age: 20-24 month-old
tissue: pool of anterior lobe prostate stroma laser capture microdissected from three mice
amplification: two round aRNA
|
| Growth protocol |
Young (4 month-old) and old (20-24 monthold) C57BL/6 male mice were obtained from the National Institutes of Aging (NIA) Rodent Colony at Harlan Sprague Dawley (Chicago, IL) and maintained in a barrier facility and cared for in accordance with approved IACUC protocols. Following shipment, mice were acclimated to a common temperature, day-night cycle, and diet for at least 12 days to minimize environmental differences. Mice from each age group were randomized for the day and time of sacrifice. Following halothane anesthesia, mice were sacrificed by cervical dislocation. Prostates were rapidly excised, immersed in OCT embedding compound (Miles Diagnostics, Elkhart, Ind., USA) and snap-frozen in liquid nitrogen and stored at -80º C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Approximately 10,000 cells were laser-capture microdissected using the Veritas LCM system (Arcturus Mountain View, CA). Total RNA from LCM experimental samples were isolated using the Arcturus PicoPure RNA Isolation kit (Molecular Devices, Sunnyvale, CA) and DNAse-treated using the RNase-Free DNase Set (Qiagen Inc, Valencia, CA) followed by two rounds of mRNA amplification with amino-allyl UTP incorporation into the second round aRNA using the Ambion MessageAmp aRNA Kit (Ambion Inc, Austin, TX), according to the manufacturer’s specifications.
|
| Label |
Cy3
|
| Label protocol |
A total of 825 ng of amplified amino-allyl aRNA from each experimental sample was labeled with Cy3 fluorescent dye (reference amino-allyl aRNA was labeled with Cy5) following Agilent suggested protocols.
|
| |
|
| |
| Hybridization protocol |
Experimental and reference probes were combined and hybridized to custom Agilent 44K whole mouse genome expression oligonucleotide microarray slides (Agilent Technologies, Inc., Santa Clara, CA) following the manufacturer’s suggested protocols.
|
| Scan protocol |
Fluorescence array images were collected for Cy3 and Cy5 using the Agilent DNA microarray scanner G2565BA (Agilent Technologies, Inc., Santa Clara, CA).
|
| Description |
pool of anterior lobe prostate stroma laser capture microdissected from three old (20-24 month-old) mice
|
| Data processing |
Agilent Feature Extraction software was used to normalize the Agilent array data using Agilent's default protocol (essentially LOWESS dye normalization on background subtracted signal values). Spots of poor quality or average intensity levels <300 were removed from further analysis.
|
| |
|
| Submission date |
Apr 27, 2010 |
| Last update date |
Dec 31, 2010 |
| Contact name |
Ilsa Coleman |
| E-mail(s) |
icoleman@fredhutch.org
|
| Phone |
206-667-1703
|
| Organization name |
Fred Hutchinson Cancer Center
|
| Department |
Human Biology
|
| Lab |
Peter Nelson
|
| Street address |
1100 Fairview Ave N, E2-112
|
| City |
Seattle |
| State/province |
WA |
| ZIP/Postal code |
98109 |
| Country |
USA |
| |
|
| Platform ID |
GPL10362 |
| Series (1) |
| GSE21542 |
The Effects of Aging on the Molecular and Cellular Composition of the Prostate Microenvironment |
|
