|
| Status |
Public on May 18, 2016 |
| Title |
22024_11 Lung |
| Sample type |
RNA |
| |
|
| Source name |
Lung
|
| Organism |
Homo sapiens |
| Characteristics |
individual id: 22024
tissue: Tumor-free lung
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Hope-fixed, paraffin-embedded tissues were cut on a microtome and transferred into microcentrifuge tubes to retrieve rolls of each specimen. Samples were subsequently deparaffinized by following protocol: 10 min. 1ml 100% xylene on a rotating device followed by centrifugation at 5000 rpm for 5 min. Supernatant was removed and step repeated. After 2nd incubation with xylene, 1ml of 100% EtOH was added and samples rotated for 10 min as well as 5 min. of centrifugation. EtOH was repeated. After xylene and EtOH incubation, the samples were dried using a vacuum centrifuge until totally dry. Deparaffinized samples were then subjected to RNA isolation with Qiagen Rneasy Mini Kit according to manufacturer´s instructions.
|
| Label |
Cy3
|
| Label protocol |
Cy3-labelling of total RNA was conducted with the Low Input Quick Amp Labelling Kit (Agilent) according to manufacturer´s instructions. Dye incorporation rate and specific yield was calculated using an NanoDrop spectrophotometer.
|
| |
|
| Hybridization protocol |
1650 ng of Cy3-labelled cRNA was fragmented according to Low Input Quick Amp Labelling Kit Protocol (Agilent) with 2xHI-RPM Hybridization Buffer for 30 min at 60°C. Hybridization on Agilent Human Whole Genome 4x44k V2 arrays was performed at 65°C for 17h under constant rotation in a hybidization oven. Washing was performed using the GE Wash buffer 1 (Agilent) at RT and Ge Wash Buffer 2 (Agilent) at 37°C for each 1 min. Washed slides were air-dried and immediately scanned.
|
| Scan protocol |
Slides were immediately scannend on an Agilent SureScan Scanner with Dye Channel =Green, 5 µm Resolution and scanning area=61 x 21.6 mm.
|
| Data processing |
Agilent Feature Extraction Software V 11.5 was used with default options (GE 1-color protocol) to extract raw data. Txt files were imported into GeneSpring software Version 13 under omission of compromised flags. Flags were accepted if at least 100 percent of samples in any 1 out of 7 conditions were detected.
|
| |
|
| Submission date |
Nov 05, 2015 |
| Last update date |
May 20, 2016 |
| Contact name |
Sebastian Marwitz |
| Organization name |
Research Center Borstel - Leibniz Lung Center
|
| Department |
Pathology
|
| Street address |
Parkallee 3a
|
| City |
Borstel |
| ZIP/Postal code |
23845 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE74706 |
Transcriptome of human NSCLC tissues |
|
