|
| Status |
Public on Jun 30, 2013 |
| Title |
A549 Hypoxic + BB2-125, biological rep2 |
| Sample type |
RNA |
| |
|
| Source name |
A549_hypoxic_treated with BB2-125
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: A549
oxygen condition: hypoxic
treated with: BB2-125
|
| Treatment protocol |
Compounds were examined under both normoxic and hypoxic conditions. Briefly, after attachment cells were treated with 1.5 mL of fresh media containing OOPs BB2-125, BB2-162, or BB2-282 at final concentrations of 10 µM. All samples contained a final concentration of 0.1% DMSO; vehicle samples were treated with cell culture media containing 0.1% DMSO. After 6 h, hypoxia was induced with GasPak EZ pouch and cells were incubated for another 42 hours. Cells were washed twice with ice-cold PBS and lysed. Total RNA was isolated with an RNeasy kit (Qiagen) according to the manufacturer’s instructions and quantified by UV absorbance. The RNA was further treated with DNase I (Ambion, DNAfree kit) to remove any remaining genomic DNA. Reverse transcription was performed with Superscript III Reverse Transcriptase (Invitrogen) as recommended by the manufacturer.
|
| Growth protocol |
A549 cells (~70% confluent) were plated in 6-well dishes (BD Falcon) at density of 150,000 cells/mL.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated with an RNeasy kit (Qiagen) according to the manufacturer’s instructions and quantified by UV absorbance. The RNA was further treated with DNase I (Ambion, DNAfree kit) to remove any remaining genomic DNA. Reverse transcription was performed with Superscript III Reverse Transcriptase (Invitrogen) as recommended by the manufacturer.
|
| Label |
biotin
|
| Label protocol |
Ambion Whole Transcript (WT) Expression Kit from Applied Biosystems used as per kit instructions. The Affymetrix Genechip WT Terminal Labeling and Hybridization kit used for Fragmentation and Labeling as per the kit instructions.
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| |
|
| Hybridization protocol |
Hybridization reaction used following the Hybridization protocol from the Affymetrix Genechip WT terminal labeling and hybridization kit (page 16). Array format/size is 169 for Human Gene 1.0 ST Array. Reaction mix per sample: DMSO- 7µl, Water- 7.3 µl, 2x Hybridization Buffer- 50 µl, Control Oligonucleotide B2- 1.7 µl, Heated 20X Eukaryotic Hybridization Controls- 5 µl, Bovin Serum Albumin - 1.0 µl, Herring Sperm DNA- 1.0 µl, total volume of mix is 73 µl. 73 µl of mix added to 27 µl (25ng/µl) of fragmented & labeled DNA, now named hybridization cocktail. The hyb. cocktail is vortexed, spun down, & transferred to a heat block for 5 minutes @ 99°C, then transferred to 45°C heat block for 5 minutes, and then spun down for 1 min @ 13,000 xg (room temp.) in Eppendorf Microcentrifuge. Chips are removed from packaging, 80 µl of hyb. cocktail (as per the 169 array format, page 17) is inserted into each chip. Samples are hybridized in Genechip Hybridization oven for 17 hours at 45°C at 60 rpm rotation.
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| Scan protocol |
After 17 hrs, The chips are removed from oven. Sample mix is removed from interior portion of chips. 85 µl of Wash Buffer A is inserted. Chips are run on Genechip Fluidics Station 450 using Fluidics Script: FS450_0007. Staining protocol used for this script is Position 1- Sape (S1) solution mix- 600 µl, Position 2- Antibody (S2) solution mix- 600 µl, and Position 3- 1x Array Holding Buffer- 800 µl. Total Run time is 1hour, 20 mins. Chips are inspected and any bubbles are removed from interior portion of chip by adding more 1x Array Holding Buffer. Chips are cleaned with a lint wipe and scanned in the Affymetrix Genechip 7G Scanner to generate CHP and CEL files.
|
| Data processing |
The data were analyzed with RMAExpress version 1.0.5 using Affymetrix default RMA expression settings with quantile normalization and summarizing using median polish.
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| |
|
| Submission date |
Jun 20, 2013 |
| Last update date |
Jun 30, 2013 |
| Contact name |
Bogdan Olenyuk |
| E-mail(s) |
bogdan@usc.edu
|
| Organization name |
University of Southern California
|
| Department |
Pharmacology and Pharmaceutical Sciences
|
| Street address |
1985 Zonal Ave. PSC B15-C
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
91089 |
| Country |
USA |
| |
|
| Platform ID |
GPL6244 |
| Series (1) |
| GSE48134 |
Designed Oligooxopiperazines as Modulators of Hypoxia-Inducible Factor Signaling |
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