|
| Status |
Public on Jun 30, 2011 |
| Title |
Immature Dendritic cells replicate 1 |
| Sample type |
mixed |
| |
|
| Channel 1 |
| Source name |
Total RNA from immature dendritic cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: immature dendritic cell
|
| Treatment protocol |
One mL of fresh medium with GM-CSF and IL-4 was additionally added to the monocyto cell cultures at day 3. Old medium was replaced by 3 mL fresh medium with GM-CSF and IL-4 at day 5. DCs were stimulated with IL-1β (10 ng/mL), IL-6 (10 ng/ml), TNF-α (10 ng/ml), and PGE2 (1 µg/mL) on day 6, and DCs were harvested on day 8 or the indicated time.
|
| Growth protocol |
Purified monocytes were cultured at 37°C in 6-well plates (1x106 cells per well) in 3 mL of AIM V medium containing human granulocyte/macrophage colony stimulating factor (GM-CSF, 100 ng/mL) and human interleukin 4 (IL-4, 20 ng/mL).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Trizol® reagent (Invitrogen) was used to extract total RNA from cells
|
| Label |
Cy3
|
| Label protocol |
Total RNA was ligated to a synthetic linker, 5’-pCU-DY547-3’. Reference DNA oligonucleotides (Invitrogen) complementary to a subset of mammalian miRNAs were combined and labeled with a ULYSIS® Alexa Fluor® 647 kit.
|
| |
|
| Channel 2 |
| Source name |
Reference DNA
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: synthetic DNA oligos corrresponding to human miRNAs
|
| Treatment protocol |
One mL of fresh medium with GM-CSF and IL-4 was additionally added to the monocyto cell cultures at day 3. Old medium was replaced by 3 mL fresh medium with GM-CSF and IL-4 at day 5. DCs were stimulated with IL-1β (10 ng/mL), IL-6 (10 ng/ml), TNF-α (10 ng/ml), and PGE2 (1 µg/mL) on day 6, and DCs were harvested on day 8 or the indicated time.
|
| Growth protocol |
Purified monocytes were cultured at 37°C in 6-well plates (1x106 cells per well) in 3 mL of AIM V medium containing human granulocyte/macrophage colony stimulating factor (GM-CSF, 100 ng/mL) and human interleukin 4 (IL-4, 20 ng/mL).
|
| Extracted molecule |
other |
| Extraction protocol |
Trizol® reagent (Invitrogen) was used to extract total RNA from cells
|
| Label |
Cy5
|
| Label protocol |
Total RNA was ligated to a synthetic linker, 5’-pCU-DY547-3’. Reference DNA oligonucleotides (Invitrogen) complementary to a subset of mammalian miRNAs were combined and labeled with a ULYSIS® Alexa Fluor® 647 kit.
|
| |
|
| |
| Hybridization protocol |
A mixture of labeled RNA and DNA was hybridized to a miRNA microarray (Thomson et al), inside a MAUI mixer in a MAUI hybridization system (BioMicro® System, Inc, Salt Lake City, UT, USA). Thomson, J.M., Parker, J., Perou, C.M., and S.M. Hammond. 2004. A custom microarray platform for analysis of microRNA gene expression. Nat. Methods 1: 47-53. Epub 2004 Sep 29.
|
| Scan protocol |
Slides were scanned with a ScanArray 5000 machine (Perkin Elmer®, Waltham, MA, USA).
|
| Description |
n/a
|
| Data processing |
BlueFuse (BlueGenome, Cambridge, UK) was used to quantify pixel intensities. GeneSpring GX 7.3.1 (Agilent Technologies) was used for data normalization.
Data processed by Bluefuse is absolute value, and from GeneSpring is normalized ratio.
|
| |
|
| Submission date |
May 06, 2010 |
| Last update date |
Jun 30, 2011 |
| Contact name |
Xiaoxiao Zhang |
| E-mail(s) |
zhang688@umn.edu
|
| Organization name |
University of Minnesota
|
| Street address |
312 Church St. SE
|
| City |
Minneapolis |
| ZIP/Postal code |
55455 |
| Country |
USA |
| |
|
| Platform ID |
GPL9517 |
| Series (1) |
| GSE21708 |
Regulation of Human Dendritic Cell Apoptosis and IL-12 Production by MiR-221and MiR-155 through targeting of p27kip1, KPC1 and SOCS1 |
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