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| Status |
Public on Feb 24, 2017 |
| Title |
Englerin A_3 h, rep. 2 |
| Sample type |
RNA |
| |
|
| Source name |
Englerin A, 3 h-treated A-498 cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A-498 (ATCC HTB-44)
tumor type: renal cell carcinoma
gender: female
treated with: ENGLERIN A for 3hrs
|
| Treatment protocol |
A498 cells were plated in complete RPMI onto 100 mm dishes at 0.9 x 10E6 cells per dish. After cells were allowed to adhere overnight, cells were refed with complete RPMI containing 0.1% DMSO (vehicle) or 100 nM englerin A and incubated for 3 hours.
|
| Growth protocol |
A498 cells were purchased from ATTC and maintained in RPMI medium supplemented with 10% FBS, 2 mM L-glutamine, and 100 units/ml penicillin/streptomycin (complete RPMI). All experiments were conducted with cells of passage number less than 25.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Cell extracts were prepared using 1 ml of TRI Reagent (Zymo Research, Irvine, CA) per dish. Total RNA was then purified from cell extracts using spin columns by Zymo Research according to the manufacturer’s directions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng total RNA using the Low Input Quick Amp Labeling Kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with a NanoDrop 2000c Spectrophotometer.
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| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >= 15.6 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarrays (G4851B-039494) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then immersed in acetonitrile and dried immediately by slowly removing the slide.
|
| Scan protocol |
Slides were scanned immediately after washing on an Agilent DNA Microarray Scanner (G2505C) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3 um, Dye channel is set to Green at 20 bit).
|
| Description |
US10130359_253949437667_S01_GE1_107_Sep09_2_4.txt
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid 039494_D_F_20140813) to obtain background subtracted and spatially detrended Processed Signal intensities. Data was uploaded into Genespring version 11.5.1 where was thresholded to 1.0, log2 transformed, quantile normalized and base line transformed using the median of all samples. Data was filtered by flags in a way that at least 75% of the samples in any of the 2 experimental groups had DETECTED flags. Then, differentially expressed genes were determined using a moderated t-test statistics with p-value 0.01 and fold change 1.5 as cutoffs.
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| |
|
| Submission date |
Aug 25, 2016 |
| Last update date |
Feb 24, 2017 |
| Contact name |
Diego Altomare |
| Organization name |
University of South Carolina
|
| Department |
Department of Drug Discovery and Biomedical Sciences
|
| Lab |
Functional Genomics Core
|
| Street address |
715 Sumter Street, Room 617
|
| City |
Columbia |
| State/province |
SC |
| ZIP/Postal code |
29208 |
| Country |
USA |
| |
|
| Platform ID |
GPL17077 |
| Series (2) |
| GSE86044 |
Gene expression analysis of renal cell carcinoma cell line A-498 after 3-hour treatment with englerin A. |
| GSE86047 |
Gene Expresion Analysis of A-498 cells after treatment with Englerin A |
|
| Relations |
| Reanalyzed by |
GSM2292543 |
| Reanalyzed by |
GSM2292551 |
