|
Status |
Public on Jan 03, 2011 |
Title |
EV4, biological rep3 |
Sample type |
RNA |
|
|
Source name |
Human rhabdomyosarcoma cell line
|
Organism |
Homo sapiens |
Characteristics |
gender: female cell type: rhabdomyosarcoma cell infection: EV71 time: 4 h
|
Growth protocol |
Human rhabdomyosarcoma cells (RD) were cultured in MEM medium with 1 mM L-glutamate, 100 units/mL penicillin, 100 £gg/mL streptomycin and 10% fetal bovine serum (Gibco) at 37¢XC, 20% O2 and 5% CO2.
|
Extracted molecule |
total RNA |
Extraction protocol |
Trizol extraction of total RNA was performed according to the manufacturer's instructions. Total RNAs extracted using TRIZOL reagent were quantified spectrophotometrically. The cDNA synthesis was carried out by using SUPERSCRIPTTM one-step RT-PCR kit (Invitrogen). For the reverse transcription step, the whole 5 ul of the resuspended RNAs were incubated for 60 min at 42oC, then 15 min at 72oC in 50 ul of reaction mixture containing 25 ul of 2x Reaction Mix (Invitrogen) and 1 ul of RT/PLANTINUM Taq Mix (Invitrogen). 28.5 ul of the cDNAs present in the 50 ul RT reaction mixture purified by Microarray Target Purification Kit (Roche Applied Science, Indianapolis, ID, USA, http://www.roche-applies-science.com) were used as templates to amplify cDNA by Microarray Target Amplification Kit. The amplified cDNAs were purified by Microarray Target Purification Kit, and complementary RNAs (cRNAs) were synthesized from 200 ng cDNA with Microarray RNA Target Synthesis Kit (Roche Applied Science).
|
Label |
biotin
|
Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 5 micrograms purified cRNA (Expression Analysis Technical Manual, 2004, Affymetrix).
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|
|
Hybridization protocol |
Following fragmentation, 10 micrograms of cRNA were hybridized for 16 hr at 45oC on Affymetrix Human Genome U133 plus 2.0 GeneChip. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
Scan protocol |
GeneChips from the hybridization experiments were read by the Affymetrix GeneChip scanner 3000.
|
Description |
The mRNA profiles from RD cells infected with/without EV71 were analyzed using Affymetrix Human Genome U133 plus 2.0 GeneChip according to the Manufacturer¡¦s protocols (Santa Clara, CA, USA, http://www.affymetrix.com).
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Data processing |
The data were processed by GeneSpring GX software version 7.3.1 GC-RMA preprocessor to use on the CEL files
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Submission date |
Mar 20, 2009 |
Last update date |
Jan 03, 2011 |
Contact name |
Sung-Liang Yu |
E-mail(s) |
slyu@ntu.edu.tw
|
Phone |
886-2-23958341
|
Organization name |
National Taiwan University
|
Department |
Clinical Laboratory Sciences and Medical Biotechnology
|
Lab |
Microarray Core Facility
|
Street address |
Jen Ai Road Section1
|
City |
Taipei |
ZIP/Postal code |
100 |
Country |
Taiwan |
|
|
Platform ID |
GPL570 |
Series (1) |
GSE15323 |
The differentially expressed genes induced by EV71 infection |
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