|
| Status |
Public on Apr 12, 2007 |
| Title |
DC_mRNA_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
mRNA-loaded dendritic cells replicate2
|
| Organism |
Homo sapiens |
| Characteristics |
Mature denritic cells pooled from 10 different normal donors, loaded with exogenously-derived mRNA, mRNA was derived from 1 of 3 different AML tumor products
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted uzing the Trizol method
|
| Label |
Biotin
|
| Label protocol |
Used the GeneChip IVT Labeling Kit (Affymetrix P/N 900449) for synthesis of Biotin-labeled cRNA. Prepared each sample reaction using all of template cDNA and incubated for 16 hours at 37 degrees C. Cleaned up and Quantified the biotin-labeled cRNA by using GeneChip® Sample Cleanup Module (Affymetrix P/N 900371). Used the NanoDrop ND-1000 spectrophotometer to quantified the cRNA and then calculated adjusted yield.
|
| |
|
| Hybridization protocol |
Used the GeneChip® Eukaryotic Hybridization Control Kit (Affymetrix P/N 900299 or 900362).
Set up reaction based on following the Human U133 Plus 2.0 chart in a 49 format array. Incubated at 99 degrees for 5 minutes then 45 degrees for 5 minutes. Pre-fill array with 250 uL of 1X hybridization Buffer and rotate in hyb oven for 10 minutes @ 45 degrees C.
1X Hybridization buffer consisted of:
8.3mL of 12X MES (Morhpolinoethanesulfonic Acid)
17.7mL of 5M NaCl
4.0mL of 0.5M EDTA
0.1mL of 10% Tween-20
19.9mL of water
50.0 mL Total
Remove 1 X pre-hyb buffer from array and inject 200 uL of Hyb cocktail mix. Rotate in hyb-oven 16-18 hours at 45 degrees C.
Add 200 uL Wash Buffer A
Wash Buffer A
300 ml of 20X SSPE
1.0 ml of 10% Tween-20
699 ml of water
1000 ml Total
Prepared SAPE solution and then prepared Antibody Solution Mix. Used Fluidics script EukGE-WS2v5_450 to run the fluidics station (Affymetrix Fluidics 450). Used Affymetrix GeneChip® Operating Software (GCOS) to run the fluidics script.
Washing and Staining:
Wash Buffer B
83.3 mL of 12X MES Stock Buffer
5.2 mL of 5M NaCl
1.0 mL of 10% Tween-20
910.5 mL of water
1000 mL Total
2X Stain Buffer
41.6 mL 12X MES Stock Buffer
92.5 mL 5 M NaCl
2.5 mL 10% Tween 20
113.3 mL Water
250 mL Total
EukGE-WS2v5 wash cycle (used for Human U133 Plus 2.0 array) was used.
|
| Scan protocol |
GCS3000 scanner
|
| Description |
mRNA loaded dendritic cells
|
| Data processing |
The software used in scanning to create the images and perform basic analysis is the Affymetrix GCOS (GeneChip® Operating Software). All the normalizations and algorithms are set within the software.
|
| |
|
| Submission date |
Mar 12, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
William K Decker |
| E-mail(s) |
wkdecker@mdanderson.org
|
| Phone |
713-563-1896
|
| Fax |
713-563-1898
|
| Organization name |
University of Texas MD Anderson Cancer Center
|
| Department |
Stem Cell Transplantation and Cellular Therapy
|
| Lab |
Cell Therapy Research Laboratory
|
| Street address |
1515 Holcombe Blvd.
|
| City |
Houston |
| State/province |
TX |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL570 |
| Series (1) |
| GSE7247 |
Dendritic Cells Compare the Similarity of Endogenous and Exogenous Antigens |
|
| Relations |
| Reanalyzed by |
GSE64985 |
| Reanalyzed by |
GSE119087 |
