|
| Status |
Public on Oct 29, 2021 |
| Title |
AR3085_01 |
| Sample type |
RNA |
| |
|
| Source name |
HEK293T, vehicle, 24 hours.
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
treatment: vehicle, 24 hours.
|
| Treatment protocol |
80% confluence 293T (HEK293T) were incubated in DMEM/F-12,Hepes without 10% fetal bovine serum (Invitrogen), 100 units/mL penicillin, and 100 μg/mL streptomycin for 24 hr. Then HEK293T were challenged with 1064 µg/cm2 calcium oxalate monohydrate (COM) or sodium oxalate (NaOx) 4mM or vehicle for 24 hours.
|
| Growth protocol |
Cell lines of human embryonic kidney cells 293T (HEK293T) were grown to confluence in DMEM/F-12,Hepes (Thermo Fisher Scientific), respectively, on plastic dishes (Falcon). The medium was supplemented with 10% fetal bovine serum (Invitrogen), 100 units/mL penicillin, and 100 μg/mL streptomycin (Thermo Fisher Scientific).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from cells using RNeasy Plus Mini Kit (QIAGEN) according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.1ug Total RNA using the Low Input Quick Amp Labeling Kit(Agilent) according to the manufacturer's instructions, followed by RNeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
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| |
|
| Hybridization protocol |
0.6ug of Cy3-labelled 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’s instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to SurePrint G3 Human GE 8x60K Microarray Ver3.0 (Agilent) 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).
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent SureScan Microarray Scanner (G2600D) using one color scan setting for 8x60k array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green PMT is set to 100%).
|
| Description |
Agilent Human 1 cDNA Microarray (G4100A) [layout A]
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 12.0.3.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Processed signal intensities were normalized by the global scaling method. A trimmed mean probe intensity was determined by removing 2% of the lower and the higher end of the probe intensities in order to calculate the scaling factor. Normalized signal intensities were then calculated from the target intensity on each array using the scaling factor, so that the trimmed mean target intensity of each array was arbitrarily set to 2500.
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| |
|
| Submission date |
Oct 27, 2021 |
| Last update date |
Oct 30, 2021 |
| Contact name |
Hidekazu Sugiura |
| E-mail(s) |
sugiura@twmu.ac.jp
|
| Organization name |
Tokyo Women’s Medical University
|
| Street address |
Kawada-cho 8-1
|
| City |
Sinjuku-ku |
| State/province |
Tokyo |
| ZIP/Postal code |
162-8666 |
| Country |
Japan |
| |
|
| Platform ID |
GPL20844 |
| Series (1) |
| GSE186676 |
Expression factors for oxalate stimulation in HEK293T cells |
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