|
| Status |
Public on Jan 31, 2009 |
| Title |
rAAVpp65N-rep3 |
| Sample type |
RNA |
| |
|
| Source name |
fibroblasts-MRC-5 cells transduced with recombinant DNA, WT PP65
|
| Organism |
Homo sapiens |
| Characteristics |
WT PP65
|
| Treatment protocol |
The MRC-5 cells were transduced in triplicate, with rAAVintA, rAAVpp65N, rAAVpp65NLSKO and incubated for 48hrs at 37C.
|
| Growth protocol |
The MRC-5 cells were grown in Dulbecco’s modified Eagle’s medium with low glucose (DMEM low) supplemented with 10% fetal bovine serum ( FBS) ( Hyclone, Utah), 2mM L-glutamine(G ), 100units/ml penicillin and 100μg/ml streptomycin ( P/S)
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was purified with RNeasy kit (Qiagen, Valencia, CA) according to the manufacturer’s instructions. ). After ribosomal RNA reduction (Invitrogen, Carlsbad, CA) to remove most of the 18S and 28S rRNAs, the GeneChip WT (whole transcript) cDNA Synthesis Kit, WT cDNA Amplification Kit, and WT Terminal Labeling Kit (Affymetrix, Inc., Santa Clara, CA) were used for target preparation. Ten µg of cRNA (antisense RNA) were added to the second-cycle cDNA reaction followed by fragmentation and Terminal Labeling process.
|
| Label |
Biotin
|
| Label protocol |
One µg of each sample was processed using the Affymetrix GeneChip Whole Transcript Sense Target Labeling Assay (https://www.affymetrix.com/support/downloads/manuals/wt_sensetarget_label_manual.pdf
|
| |
|
| Hybridization protocol |
The hybridization cocktails containing 2.5 ug of the fragmented, end-labeled cDNA were applied to the GeneChip® Human Gene 1.0 ST arrays. Hybridization was performed for 16 hours, and the arrays were washed and stained with the GeneChip Fluidics Station 450 using FS450_0007 script.
|
| Scan protocol |
Arrays were scanned using the Affymetrix GCS 3000 7G and GeneChip Operating Software v. 1.4 to produce CEL intensity files.
|
| Description |
MRC-5 cells transduced with rAAV wide type CMV PP65
|
| Data processing |
Data were background corrected with GC bin, quantile normalized and summarized by interPLIER algorithm with Affymetrix Expression Console.
|
| |
|
| Submission date |
Jan 08, 2009 |
| Last update date |
Jan 09, 2009 |
| Contact name |
Xiwei Wu |
| E-mail(s) |
xwu@coh.org
|
| Organization name |
City of Hope National Medical Center
|
| Department |
Computational and Quantitative Medicine
|
| Street address |
1500 E. Duarte Rd.
|
| City |
Duarte |
| State/province |
CA |
| ZIP/Postal code |
91010 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE14347 |
Knock-out of the nuclear localization in pp65 protein of Cytomegalovirus: biologic and immunologic effects |
|
