|
| Status |
Public on Apr 21, 2008 |
| Title |
CD 34- vs. CD 34+ Cy3: CD 34+ Cy5: CD34- |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
CD 34+
|
| Organism |
Mus musculus |
| Characteristics |
treated
|
| Treatment protocol |
Keratinocytes were harvested from adult mouse skin (7 weeks of age) following digestion in 0.25% trypsin for 2 hours at 32 C. Single cell preparations were made after epidermal cells were scraped into fresh media (SMEM supplemented with 10% fetal bovine serum and 1X gentamicin). Cells were stained with antibodies to CD34 (rat anti-mouse CD34 RAM clone and rat anti-human alpha-6 integrin) and sorted into alpha6+CD34+ (stem and progenitor cells) and alpha6+CD34- (basal cells) using fluorescence activated cell sorting (FACS).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The CD34+ and CD34- cells were pelleted and lysed in 1 ml lysis buffer with beta-mercaptoethanol (Qiagen Rnasesy kit), then total RNA extracted following manufacturer’s protocol.
|
| Label |
Cy3
|
| Label protocol |
Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 0.5µg of total RNA, Cy3 or Cy5 labeled cRNA was produced according to manufacturer's protocol.
|
| |
|
| Channel 2 |
| Source name |
CD34-
|
| Organism |
Mus musculus |
| Characteristics |
control
|
| Treatment protocol |
Keratinocytes were harvested from adult mouse skin (7 weeks of age) following digestion in 0.25% trypsin for 2 hours at 32 C. Single cell preparations were made after epidermal cells were scraped into fresh media (SMEM supplemented with 10% fetal bovine serum and 1X gentamicin). Cells were stained with antibodies to CD34 (rat anti-mouse CD34 RAM clone and rat anti-human alpha-6 integrin) and sorted into alpha6+CD34+ (stem and progenitor cells) and alpha6+CD34- (basal cells) using fluorescence activated cell sorting (FACS).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The CD34+ and CD34- cells were pelleted and lysed in 1 ml lysis buffer with beta-mercaptoethanol (Qiagen Rnasesy kit), then total RNA extracted following manufacturer’s protocol.
|
| Label |
Cy5
|
| Label protocol |
Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 0.5µg of total RNA, Cy3 or Cy5 labeled cRNA was 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 |
Chips were scanned with an Agilent Scanner and processed with the Agilent Feature Extraction Software (v7.5).
|
| Description |
Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng 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 (v7.5).
|
| Data processing |
Agilent Feature Extraction protocol for 44K arrays, log2ratio Treated/Control
|
| |
|
| Submission date |
May 02, 2007 |
| Last update date |
Apr 21, 2008 |
| 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 |
GPL891 |
| Series (1) |
| GSE7690 |
Comprehensive Microarray Transcriptome Profiling of CD34-Enriched Mouse Keratinocyte Stem Cells |
|
