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Sample GSM1286156 Query DataSets for GSM1286156
Status Public on Jun 20, 2014
Title Ataxin-2 Overexpression_48hr_rep1
Sample type RNA
Source name HEK293T, Ataxin-2 Overexpression, replicate 1
Organism Homo sapiens
Characteristics cell: HEK293T
condition: Halo-Ataxin-2 overexpression
Treatment protocol For the knockdown experiment, either Ataxin-2 siRNA or Control siRNA was transfected at the final concentration of 10 nM into 2 x 10^4 HEK293T cells using Lipofectamine RNAiMAX (Life Technologies). For the overexpression experiment, 8 μg of either pFN21A-Halo-Ataxin-2 or pFN21A-Halo was transfected into HEK293T cells that were 90% confluent in 6-cm dish using Lipofectamine 2000 (Life Technologies). Twenty-four hours after transfection, the cell culture medium was replaced with fresh medium, and the cells were incubated for additional 24 hrs at 37 ºC in a humidified incubator with 5% CO2
Growth protocol HEK293T cells were grown in DMEM medium with 10% FBS in 6-cm dish at 37 ºC in a humidified incubator with 5% CO2
Extracted molecule total RNA
Extraction protocol Total RNA was extracted using TRIZOL reagent followed by further purification with PureLink RNA Mini Kit (Life Technologies) following the manufacturer's recommendations. The protocol includes an on-column DNase digestion. RNA was quantified using a NanoDrop-1000 spectrophotometer and quality was monitored with the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
Label Cy3
Label protocol Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1 ug RNA using Low Input Quick Amp labeling kit, one-color (Agilent) according to the manufacturer's instructions, followed by RNAeasy mini column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
Hybridization protocol 0.6 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE microarray kit 8x60k Ver2.0 (G4851B) 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 dried immediately by brief centrifugation.
Scan protocol Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2539A) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
Description Gene expression 48 hr after The transfection of Halo-Ataxin-2 expressiion construct
Data processing The scanned images were analyzed with GeneSpring GX 12.6.0 (Agilent) using default parameters (Grit temptate 039494_D_F_20120628, FE protocol GE1_1010_Sep10, QC Metric GE1_QCMT_Sep10).
Submission date Dec 10, 2013
Last update date Jun 20, 2014
Contact name Yukio Kawahara
Phone +81-6-6879-3827
Organization name Osaka University
Department Graduate School of Medicine
Lab Laboratory of RNA Function
Street address 2-2 Yamada-oka
City Suita
State/province Osaka
ZIP/Postal code 565-0871
Country Japan
Platform ID GPL17077
Series (1)
GSE53189 Analysis of the expression of Ataxin-2-target genes

Data table header descriptions
VALUE Normalized signal intensity

Data table
A_23_P117082 4.203
A_33_P3246448 null
A_33_P3318220 null
A_33_P3236322 null
A_33_P3319925 null
A_21_P0000509 9.973
A_21_P0000744 1.886
A_24_P215804 -0.809
A_23_P110167 2.524
A_33_P3211513 0.846
A_23_P103349 null
A_32_P61480 null
A_33_P3788124 null
A_33_P3414202 -1.353
A_33_P3316686 0.067
A_33_P3300975 3.783
A_33_P3263061 3.34
A_33_P3261373 null
A_24_P278460 0.578
A_21_P0013109 null

Total number of rows: 50599

Table truncated, full table size 947 Kbytes.

Supplementary file Size Download File type/resource
GSM1286156_Target_OE-1.txt.gz 12.4 Mb (ftp)(http) TXT
Processed data included within Sample table

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