|
| Status |
Public on Sep 09, 2014 |
| Title |
4-act R69 [miRNA] |
| Sample type |
RNA |
| |
|
| Source name |
NK cells_act R69
|
| Organism |
Homo sapiens |
| Characteristics |
donor id: donor 4
activated by: peripheral blood lymphocytes (PBLs)
cell type: EBV-target cell (R69)
cell subtype: purified NK cells
molecule subtype: miRNA
|
| 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 |
We used 400ng of each sample to join a tail of Poly (A) with a PolyA Polymerase and then labeled with biotin following the protocol of FlashTag Biotin HSR RNA Labeling Kit for Affymetrix GeneChip miRNA arrays (Genisphere®). Just as indicated by the protocol, as a first step, RNA was added to a mixture of the starting Spike RNA controls included in the kit to allow quality control after hybridization to verify that the processing had occurred as expected.
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| |
|
| Hybridization protocol |
Following recommendations of Genisphere, we prepared a mixture of hybridization, which included Eukaryotic Hybridization Controls 20x and control oligonucleotide B2 (Affymetrix). Development of chips took place in an Affymetrix Fluidics Station, using the Hybridization, Wash and Stain kit from Affymetrix.
|
| Scan protocol |
GeneChips were scanned using the Scanner 3000 7G (Affymetrix)
|
| Description |
SAMPLE 16
11SE249_(miRNA-2_0).CEL
|
| Data processing |
Affymetrix GeneChip Command Console Software v3.0 (AGCC 3.0, Affymetrix ®) and the miRNA QC Tool Software (Affymetrix ®) were used for processing of chips and results.
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).
Th 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 |
GPL14613 |
| Series (2) |
| GSE50839 |
Expression data from activated NK cells [miRNA] |
| GSE50840 |
Expression data from activated NK cells |
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