|
| Status |
Public on May 11, 2012 |
| Title |
Nrest_3A |
| Sample type |
RNA |
| |
|
| Source name |
Murine FACS-sorted naïve (CD44lo) CD8 T cells cultured 4hrs ex vivo media only
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6N
gender: Female
cell type: CD8+Thy1.2+CD44lo
|
| Treatment protocol |
FACS-purified naïve (CD44lo) and memory phenotype (CD44hi) CD8+ T cells were cultured for four hours in media alone (Iscove’s DMEM + 8% FBS (HI) + 100U/mL Pennicillin + 100ug/mL Streptomycin + 2mM L-glutamine + 50uM β-mercaptoethanol) or in media with PMA (10ng/mL) and ionomycin (500ng/mL).
|
| Growth protocol |
n/a
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Following 4hr culture, cells were lysed in TRIzol reagent followed by phenol+chloroform extraction and purification using RNEasy mini columns (QIAGEN).
|
| Label |
Cy3
|
| Label protocol |
Approximately 200 ng of total RNA was amplified and labeled with Cy3 using the Low RNA Input Linear Amp Kit PLUS, One-Color (Agilent Technologies, CA). This labeling reaction produces 0.25 – 2.0 µg of Cy3-labeled cRNA (anti-sense), by first Converting mRNA primed with an oligo (d)T-T7 primer into dsDNA with MMLV-RT and then amplifying the sample using T7 RNA Polymerase in the presence of Cy3-CTP.
|
| |
|
| Hybridization protocol |
After purification, 1.65 μg of cRNA was fragmented and hybridized to the array for 17 hr. at 65°C.
|
| Scan protocol |
Microarray slides were hybridized overnight, then washed and scanned with Agilent G2565BA Microarray Scanner. Images were analyzed with Feature Extraction 9.5 (Agilent Technologies, CA).
|
| Description |
Murine FACS-sorted naïve (CD44lo) CD8 T cells cultured 4hrs ex vivo media only
|
| Data processing |
Mean foreground intensities were obtained for each spot and imported into the mathematical software package “R”, which was used for all data input, diagnostic plots, normalization and quality checking steps of the analysis process using scripts developed in-house by Peter White specifically for this analysis. In outline, the Cy3 (green) intensities were not background corrected (this has been shown to only introduce noise), and corrected for the scanner offset (40 was subtracted for each intensity). The dataset was filtered to remove positive control elements and any elements that had been flagged as bad. Using the negative controls on the arrays, the background threshold was determined and all values less than this value were set to the threshold value. Finally, the data was normalized using the Quantile Normalization package in “R” (Bolstad et al., 2003).
|
| |
|
| Submission date |
May 18, 2009 |
| Last update date |
May 11, 2012 |
| Contact name |
Joanna DiSpirito |
| E-mail(s) |
hshen@mail.med.upenn.edu, rdo2@mail.med.upenn.edu
|
| Organization name |
University of Pennsylvania
|
| Department |
Microbiology
|
| Lab |
Hao Shen, PhD
|
| Street address |
3610 Hamilton Walk, University of Pennsylvania
|
| City |
Philadelphia |
| State/province |
PA |
| ZIP/Postal code |
19104 |
| Country |
USA |
| |
|
| Platform ID |
GPL4134 |
| Series (1) |
| GSE16145 |
Expression analysis of resting and stimulated naïve and MP CD8 T cells |
|
