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| Status |
Public on Sep 09, 2014 |
| Title |
1-naive |
| Sample type |
RNA |
| |
|
| Source name |
NK cells_naive
|
| Organism |
Homo sapiens |
| Characteristics |
donor id: donor 1
cell type: peripheral blood lymphocytes
activated by: none(naïve control)
cell subtype: purified NK cells
|
| Treatment protocol |
PBLs from 4 different donors were activated by 100 U/ml IL-2; K562+IL-2 or R69 cells. After 5 days we obtained RNA and miRNA from naïve NK cells or from NK cells activated with the above mentioned stimuli. The 16 RNA samples were used to generate cDNA libraries that were hybridized on Human Gene 1.1ST arrays (Affymetrix) and analyzed with the Affymetrix Gene Chip Command Console Software v3.0 (AGCC 3.0, Affymetrix®) and the Expression Console Software v1.1 (Affymetrix®).
|
| Growth protocol |
5 days RPMI 5%CO2, 37 C, 1 million/ml
|
| Extracted molecule |
total RNA |
| Extraction protocol |
microRNA were isolated from enriched ex vivo or activated NK cells (more than 90% purity) by using the mirVana miRNA (Invitrogen) Isolation Kit
|
| Label |
biotin
|
| Label protocol |
cDNA was synthesized from total RNA and, after fragmentation, hybridized with the chips, following the protocol proposed by Affymetrix. Specifically, 16 total RNA samples were used to generate cDNA with the Ambion® WT Expression kit. The main steps of the protocol were: synthesis of cDNA with random oligonucleotides including the T7 promoter, transcription in vitro; new synthesis of cDNA from the resulting cRNA from above, including dUTP. The obtained cDNA was fragmented using UDG (uracil DNA glycosylase) and APE1 (apurinic/apyrimidinic endonuclease1) and labeled with terminal transferase using the terminal WT Labeling kit.
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| |
|
| Hybridization protocol |
For the preparation of mixtures of hybridization, washing, and detection of the arrays, GeneTitan Hybridization, Wash and Stain kit for WT Array Plates were used. Hybridization, developing, washing and scanning of 1.1STHuman Gene arrays was performed on an automated GeneTitan System, as recommended by Affymetrix.
|
| Scan protocol |
GeneChips were scanned using the Gene Titan System.
|
| Description |
SAMPLE 1
|
| Data processing |
Software used for the processing of the chips and the results were the Affymetrix GeneChip Command Console Software v3.0 (AGCC 3.0, Affymetrix®) and the Expression Console Software v1.1 (Affymetrix®).
We used the software dChip (www.dchip.org) to determine the existence of cross-hybridization and image contamination. We applied the program Expression Console ofAffymetrix (Gene arrays) and Partek Genomics Suite (miRNA arrays) to obtain the RLE (Relative Log Expression), in which the expression of each probe is normalized with an array of reference built with the average of all arrays for each probe set. The intensity values for each probe were processed and normalized by Robust Multichip Average (RMA). We eliminated sequences with intensities close to the background and, after normalization, those ones that did not change during the experiment in order to reduce the number of genes and the false positives. For the Gene Array we finally obtained a work list of 8711 sequences and for the miRNA we obtained 4581 (selection of all human miRNA present in the microarray).
The softwares consider as present all p-values under 0.06.
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| |
|
| Submission date |
Sep 12, 2013 |
| Last update date |
Sep 09, 2014 |
| Contact name |
martin villalba |
| E-mail(s) |
martin.villalba@inserm.fr
|
| Phone |
33467330465
|
| Organization name |
Institut for Research in Biotherapy of Montpellier
|
| Department |
INSERM U1040
|
| Street address |
CHU Montpellier Hôpital Saint-Eloi 80, av. Augustin Fliche.
|
| City |
montpellier |
| ZIP/Postal code |
34295 |
| Country |
France |
| |
|
| Platform ID |
GPL11532 |
| Series (2) |
| GSE50838 |
Expression data from activated NK cells [RNA] |
| GSE50840 |
Expression data from activated NK cells |
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