|
| Status |
Public on Jun 23, 2016 |
| Title |
PFC No. 1_linc-9432 knockdown |
| Sample type |
RNA |
| |
|
| Source name |
Pterygium fibroblast cell, knocked down, 48 h
|
| Organism |
Homo sapiens |
| Characteristics |
treatment: Linc-9432 knockdown
treatment: 48 h
|
| Treatment protocol |
21 nM pooled siRNA (Ambion, Life Technologies) or non-specific control oligonucleotides are added to cultured fibroblast cells for 48 hours
|
| Growth protocol |
Cultured primary pterygium fibroblast cells are cultured in DMEM/F-12 with 10% FBS
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the RNAeasy mini kit (Qiagen) according to manufacturer's protocol
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 1 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
600 ng 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 25 uL containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 human GE 8x60K microarray 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 High Resolution Microarray scanner 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 after treatment with siRNA targeting linc-9432 for 48 h
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 9.1 (Agilent) using default parameters (protocol GE1-v1_91 and Grid: 012391_D_20060331) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Jun 22, 2016 |
| Last update date |
Jun 23, 2016 |
| Contact name |
Wanwen Lan |
| Organization name |
Singapore Eye Research Institute
|
| Street address |
9 Hospital Drive SON Blk C #02-03
|
| City |
Singapore |
| ZIP/Postal code |
169612 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL21185 |
| Series (1) |
| GSE83628 |
Gene expression changes with knock-down of linc-9432 |
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