|
| Status |
Public on Mar 31, 2023 |
| Title |
Donor_3_Periphery |
| Sample type |
RNA |
| |
|
| Source name |
donor3, retina, peripheral region
|
| Organism |
Homo sapiens |
| Characteristics |
disease: retinoblastoma
tissue: retina
gender: female
age in month: 6m
|
| Treatment protocol |
Donor eyes were obtained from 3 patients (age 4-17 months) with retinoblastoma after enucleation. The tumor-free neural retina was immediately dissociated from the eye with forceps and surgical scissors. The macula region and peripheral retina were dissected from the neural retina using forceps and scissors and directly transferred to a tube containing RNA extraction solution.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) in accordance with the manufacturer’s instructions. The concentration and purity of the isolated RNA was evaluated using a NanoDrop LITE (Thermo Fisher Scientific, Waltham, MA, USA). The integrity of the RNA samples was analyzed on the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
|
| Label |
Cy3
|
| Label protocol |
Total RNAs (50 ng) extracted from the macular and peripheral regions of retina were used to prepare Cy3-labelled target cRNA using the Low Input Quick Amp Labeling Kit (Agilent Technologies) followed by RNeasy column purification (QIAGEN).
|
| |
|
| Hybridization protocol |
0.6 µg of Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 µl containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturer’s instructions. On completion of the fragmentation reaction, 25 µl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Sureprint G3 Human Gene Expression Microarrays 8 × 60 K v2 (Agilent Technologies) for 17 hours at 65°C in a hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with Gene Expression Wash Buffer 1 (Agilent Technologies) and 1 minute at 37°C with Gene Expression Wash Buffer 2 (Agilent Technologies), then air-dried immediately.
|
| Scan protocol |
Slides were scanned on an Agilent G2505C microarray scanner at 100% PMT and 3 um resolution.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent Technologies) using default parameters (protocol GE1_107_Sep09 and Grid: 039494_D_F_20120411) to obtain background subtracted and spatially detrended Processed Signal intensities. Normalized signal intensity (75th percentile shift normalized) calculated from the processed signal after Feature Extraction using the GeneSpring software version 12.1 (Agilent).
|
| |
|
| Submission date |
Jan 27, 2020 |
| Last update date |
Mar 31, 2023 |
| Contact name |
Taku Tanaka |
| Phone |
+81-3-3416-0181
|
| Organization name |
National Center for Child Health and Development
|
| Department |
Ophthalmology
|
| Lab |
Visual Science
|
| Street address |
2-10-1 Okura, Setagaya
|
| City |
Tokyo |
| ZIP/Postal code |
157-8535 |
| Country |
Japan |
| |
|
| Platform ID |
GPL16699 |
| Series (1) |
| GSE144300 |
Transcriptome analysis of human maturing macula |
|
