|
| Status |
Public on Mar 07, 2009 |
| Title |
CMS |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Central memory T cells (TCM) stimulated by incubation with anti-CD3 and anti-CD28 Ab.
|
| Organism |
Homo sapiens |
| Characteristics |
Blood was collected from healthy human donors, PBMCs were isolated from blood using Ficoll gradients. Naive and memory CD8 T cells were then enriched by removing other types of cells through incubation with a panel of mouse mAbs against CD4, CD19, CD11b, CD14, CD16, MHC class II, erythrocytes, platelets, and CD45RO (for the enrichment of naïve cells) or CD45RA (for that of memory cells). Ab-bound cells were subsequently removed by incubation with anti-mouse IgG-conjugated magnetic beads (Qiagen, Valencia, CA). These enriched naïve and memory CD8 T cells were further purified into CD8+CD45RA+CD62L+ naïve T cells, CD8+CD45RA-CD62L+ central memory T cells (TCM), and CD8+CD45RA-CD62L- effector memory T cells (TEM) by a cell sorter (MoFlo; Dako Cytomation, Carpentaria, CA).
|
| Treatment protocol |
Sorted naïve, TCM, and TEM CD8 T cells were either used right away or incubated with anti-CD3 and anti-CD28 Ab (anti-CD3/CD28) coupled magnetic beads (Invitrogen) at the cell:bead ratio of 1:1 for 16 hours in RPMI-1640 with 10% Fetal bovine serum and penicillin (10 U/ml)/streptomycin (10 ug/ml) (Invitrogen). The freshly isolated and 16 hr stimulated cells from several donors as a pool were used for gene expression microarray analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from freshly isolated (0Hr) and stimulated (16 hours) naïve, TCM and TEM CD8 T cells (by STAT60) from 12 different healthy adult donors to form 4 RNA pools. The quality and quantity of total RNA were analyzed by an Agilent Bioanalyzer (Agilent Technologies, Palo Alto, CA). Three pools of RNA were used in the microarray experiment.
|
| Label |
Cy3
|
| Label protocol |
Total RNA (500 ng) was reverse transcribed to make double stranded cDNA using a T7 containing oligo-dT primer and the Agilent low RNA Input Linear Amplification Kit. This was followed by cRNA synthesis with T7 RNA polymerase in the presence of 0.3 mM cyanine 3-CTP (Cy3), from the same kit. The labeled probes were cleaned up and concentrated with Qiagen's RNeasy mini spin columns. The quantity of amplified cRNA was measured by a NanoDrop small volume spectrophotometer.
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| |
|
| Channel 2 |
| Source name |
Stratagene’s Universal Human Reference RNA (Stratagene, La Jolla, CA).
|
| Organism |
Homo sapiens |
| Characteristics |
Stratagene’s Universal Human Reference RNA is composed of total RNA from 10 human cell lines.
|
| Treatment protocol |
Equal quantities of DNase-treated total RNA from each of the 10 cell lines were pooled to make the Universal Human Reference RNA.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Unknown
|
| Label |
Cy5
|
| Label protocol |
Total RNA (1200 ng) was reverse transcribed to make double stranded cDNA using a T7 containing oligo-dT primer and the Agilent low RNA Input Linear Amplification Kit. This was followed by cRNA synthesis with T7 RNA polymerase in the presence of 0.3 mM cyanine 5-CTP (Cy5), from the same kit. The labeled probe was cleaned up and concentrated with Qiagen's RNeasy mini spin columns. The quantity of amplified cRNA was measured by a NanoDrop small volume spectrophotometer.
|
| |
|
| |
| Hybridization protocol |
3 unique biological replicates of each cell type and treatment protocol were hybed to human 4X44K arrays. In short, 825ng of each of the Cy3 sample probes and Cy5 control probes were combined with 10X Blocking Agent, 25X Fragmentation Buffer and nuclease free water to 55ul and then denatured at 60°C for 30 minutes. Samples were then mixed with 55ul of Hi-RPM hybridization buffer and applied to the Human 4X44K whole genome oligo arrays using Sure-Hyb chambers and hybridized for 17 hours in a rotating chamber at 65°C. Slides were washed the following day using the Agilent wash buffers, twice at room temperature in buffer#1 and once at ~31°C in buffer #2, then dried using a stream of nitrogen and scanned.
|
| Scan protocol |
Arrays were scanned at a 5um resolution using the extended dynamic range protocol on an upgraded Agilent microarray scanner, model G2565AA.
|
| Description |
Central memory T cells (TCM) stimulated by incubation with anti-CD3 and anti-CD28 Ab.
|
| Data processing |
Data from both the Cy-3 and the Cy5 channels was extracted using the Agilent Feature Extractor Software, Version 7.5. The normalization consisted of a log10 transformation of both channels and the Cy-3 channels were normalized to the intensity of the Cy-5 control channel ( log10cy3 - log10cy5) + mean log10cy5 signals. The results from 3 biological replicates were then averaged to give the VALUE below.
|
| |
|
| Submission date |
Jan 14, 2009 |
| Last update date |
Jun 22, 2020 |
| Contact name |
Supriyo De |
| Organization name |
NIA-IRP, NIH
|
| Department |
Laboratory of Genetics and Genomics
|
| Lab |
Computational Biology & Genomics Core
|
| Street address |
251 Bayview Blvd
|
| City |
Baltimore |
| State/province |
Maryland |
| ZIP/Postal code |
21224 |
| Country |
USA |
| |
|
| Platform ID |
GPL6480 |
| Series (1) |
| GSE14422 |
Histone methylations and gene expression in naïve, central (TCM) and effector (TEM) memory CD8 T cells. |
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