|
| Status |
Public on Apr 20, 2012 |
| Title |
B Cell 2 |
| Sample type |
RNA |
| |
|
| Source name |
Purified B Cells from PBMCs
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: B Cell
status: uninfected
|
| Treatment protocol |
The cells were washed once in PBS and then re-suspended in fresh medium.
|
| Growth protocol |
Human peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll purification (Histopaque-1077 column, Sigma) of buffy coats from normal donors (Carolina Red Cross) and kept in RPMI 1640 medium (Invitrogen) supplemented with 15% Fetal Bovine serum (100-500 Gemini Bio Product), 100 U/ml Penicillin, and 2mM L-Glutamine (G6784, Invitrogen). B cells were separated using the Human B Lymphocyte Enrichment set-DM (Imag, BD) as recommended by the manufacturer. PBMCs were infected with limiting amounts of B95-8 virus for 1h at 37°C (0.4-12 μl EBV B95-8 Z-HT supernatant/1x106 PBMC) in the presence of Cyclosporine A (0.5 μg/ml). The cells were washed once in PBS and then re-suspended in fresh medium. For each condition of infection, 105 cells were plated in to 96-well plate (in total 10 wells) to grow out as LCLs. Microarray samples were generated from monoclonal LCLs produced from the lowest amount of virus used For the purification of EBV-positive and proliferating B cells, 1.5x108 PBMC were stained with 4 μM 6-carboxyfluorescein succinimidyl ester (CFSE) for 3 minutes and washed three times in washing buffer (PBS + 5% fetal bovine serum). Stained PBMC (106/ml) were infected with 25 μl/1x106PBMC of B95-8 virus as already described.The cells were washed once in PBS and then re-suspended in fresh medium. At 6 days post-infection, CFSE-labeled PBMCs were washed in washing buffer and stained with APC-labeled α-CD19 (555415-BD Biosciences) (2μl/3x105) for 30 minutes on ice. After 3 washes in washing buffer, CD19+/CFSElow cells were sorted on a FACSVantage cell sorter flow cytometer.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
mir-Vana miRNA isolation kit (Ambion) according to the manufacturer’s instructions
|
| Label |
Hy5
|
| Label protocol |
Control (Human lymph node RNA from Ambion) and test miRNA was labeled with Hy3 and Hy5, respectively, and mixed with spike-in control miRNA's.
|
| |
|
| Hybridization protocol |
Labeled miRNAs were hybridized to a miRNA microarray (Exiqon V.10) at 65 degrees Celcius for 16 hours in a nitrogen environment.
|
| Scan protocol |
The microarray slides were scanned using the GenePix 4100 scanner (Axon Instruments).
|
| Description |
miRNA microarray expression
|
| Data processing |
The quantified signals were normalized using the global Lowess algorithm, using Genespring (Agilent Technologies, Santa Clara, CA) software. The intensity values for multiple spots were averaged and the normalized values were log2-transformed. Differentially expressed microRNAs (false discovery rate<5%) were identified using SAM that were differentially expressed among the 3 groups (resting B cell, proliferating B cells and LCLs).
|
| |
|
| Submission date |
Mar 29, 2012 |
| Last update date |
Nov 25, 2024 |
| Contact name |
Dereje Jima |
| E-mail(s) |
[email protected]
|
| Organization name |
NCSU
|
| Department |
CHHE
|
| Street address |
850 Main Campus Drive
|
| City |
RALEIGH |
| State/province |
NC |
| ZIP/Postal code |
27606 |
| Country |
USA |
| |
|
| Platform ID |
GPL7722 |
| Series (1) |
| GSE36926 |
The Epstein-Barr virus induced tumor suppressor miR-34a is growth promoting in EBV-infected B cells |
|
