|
| Status |
Public on Feb 25, 2012 |
| Title |
Naïve CD4 no activation rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
Unactivated naïve CD4 T cell
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: CD4+CD45RA+ T cells
treatment: None
|
| Treatment protocol |
For microarray analysis, CD4+CD45RA+ T cells were isolated on a BD FACSAria II cell sorter (BD Biosciences, San Jose, CA) using anti-CD4 (BD, clone SK3) and CD45RA (BD, clone HI100). Cells with >99% purity were seeded at a density of 1x106 cells/mL of RMPI-1640 supplemented with 5% human heat-inactivated pooled AB serum (Valley Biomedical, Winchester, VA). Cells were activated with latex beads conjugated to control antibody (BD, clone MPC-11) or antibodies specific for anti-CD3 (clone OKT3) or to both anti-CD3 and anti-CD28 (clone 9.3).
|
| Growth protocol |
Cells were cultured in RPMI + 5% FCS from the time of isolation until RNA harvest
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA samples for microarray were prepared with Trizol (Invitrogen, Carlsbad CA) according to the manufacturer’s instructions. RNA integrity was confirmed with a Bioanalyzer 2100 (Agilent, Santa Clara CA); only samples with a RIN ≥7.5 were processed further.
|
| Label |
hy5
|
| Label protocol |
Total RNA was labeled with Cy5/Cy3 using miRCURY LNA microRNA array Power Labeling kit (Exiqon) according to the manufacturer's instructions.
|
| |
|
| Channel 2 |
| Source name |
Unactivated PBMC
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: PBMC
treatment: None
|
| Treatment protocol |
For microarray analysis, CD4+CD45RA+ T cells were isolated on a BD FACSAria II cell sorter (BD Biosciences, San Jose, CA) using anti-CD4 (BD, clone SK3) and CD45RA (BD, clone HI100). Cells with >99% purity were seeded at a density of 1x106 cells/mL of RMPI-1640 supplemented with 5% human heat-inactivated pooled AB serum (Valley Biomedical, Winchester, VA). Cells were activated with latex beads conjugated to control antibody (BD, clone MPC-11) or antibodies specific for anti-CD3 (clone OKT3) or to both anti-CD3 and anti-CD28 (clone 9.3).
|
| Growth protocol |
Cells were cultured in RPMI + 5% FCS from the time of isolation until RNA harvest
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA samples for microarray were prepared with Trizol (Invitrogen, Carlsbad CA) according to the manufacturer’s instructions. RNA integrity was confirmed with a Bioanalyzer 2100 (Agilent, Santa Clara CA); only samples with a RIN ≥7.5 were processed further.
|
| Label |
hy3
|
| Label protocol |
Total RNA was labeled with Cy5/Cy3 using miRCURY LNA microRNA array Power Labeling kit (Exiqon) according to the manufacturer's instructions.
|
| |
|
| |
| Hybridization protocol |
The SureHyb chamber kit and Gasket slide kit (Agilent) were used to hybridize the labeled RNA for 18 h at 56°C to Exiqon miRCURY LNA miRNA array V.11.0, lot# 31043.03 according to the manufacturer's instructions.
|
| Scan protocol |
Arrays were scanned on Axon GenePix 4000B scanner, and GPR files containing fluorescent ratios (sample/control or vice versa ) were generated using GenePix Pro 6.0 software
|
| Data processing |
LOWESS normalized, background subtracted data obtained from log2 of processed Red signal/processed Green signal. GeneSpring software was used.
|
| |
|
| Submission date |
Feb 24, 2012 |
| Last update date |
Feb 25, 2012 |
| Contact name |
Dimitri de Kouchkovsky |
| Organization name |
UCSF
|
| Department |
Diabetes Center
|
| Lab |
Bluestone
|
| Street address |
513 Parnassus Ave HSW 1102 Box 0540
|
| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143 |
| Country |
USA |
| |
|
| Platform ID |
GPL7723 |
| Series (1) |
| GSE36071 |
Effect of costimulation on microRNAs levels in human naïve CD4 T cells |
|
