|
| Status |
Public on Jun 20, 2014 |
| Title |
Control Overexpression_48hr_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
HEK293T, Control Overexpression, replicate 2
|
| Organism |
Homo sapiens |
| Characteristics |
cell: HEK293T
condition: Halo 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-tag 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 |
| E-mail(s) |
ykawahara@rna.med.osaka-u.ac.jp
|
| 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 |
|
