|
| Status |
Public on Jan 01, 2015 |
| Title |
Adrenocortical adenoma clincal sample 3 |
| Sample type |
RNA |
| |
|
| Source name |
ACA patient sample 3
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Adrenocortical adenoma
source: Anonymous patient sample
|
| Treatment protocol |
Samples were obtained during surgery and snap frozen in liquid nitrogen and stored at -80 C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from approximately 30 mg of fresh frozen tissue using a Qiazol protocol (RNeasy Mini Kit, Qiagen, Hilden, Germany) in accordance with the manufacturer’s instructions. RNA quality was assessed using the Agilent 2100 Bioanalyser (Agilent Technologies, Englewood, CO, USA) with a minimum RNA integrity number (RIN) of seven required.
|
| Label |
Cy3
|
| Label protocol |
Sample labeling and array hybridization were performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology). Then, each sample was amplified and transcribed into fluorescent cRNA along the entire length of the transcripts without 3’ bias utilizing a random priming method. The labeled cRNAs were purified by RNeasy Mini Kit (Qiagen). The concentration and specific activity of the labeled cRNAs (pmol Cy3/μg cRNA) were measured by NanoDrop ND-1000.
|
| |
|
| Hybridization protocol |
1 μg of each labeled cRNA was fragmented by adding 5 μl 10 × Blocking Agent and 1 μl of 25 × Fragmentation Buffer, then heated the mixture at 60 °C for 30 min, finally 25 μl 2 × GE Hybridization buffer was added to dilute the labeled cRNA. 50 μl of hybridization solution was dispensed into the gasket slide and assembled to the LncRNA expression microarray slide. The slides were incubated for 17 hours at 65°C in an Agilent Hybridization Oven.
|
| Scan protocol |
The hybridized arrays were washed, fixed and scanned with using the Agilent DNA Microarray Scanner (part number G2505C).
|
| Data processing |
Agilent Feature Extraction software (version 11.0.1.1) was used to analyze acquired array images. Quantile normalization and subsequent data processing were performed with using the GeneSpring GX v12.0 software package (Agilent Technologies). After quantile normalization of the raw data, LncRNAs and mRNAs that at least 15 out of 23 samples. Differentially expressed LncRNAs and mRNAs with statistical significance between the two groups were identified through Volcano Plot filtering. Hierarchical Clustering was performed using the Agilent GeneSpring GX software (version 12.0). GO analysis and Pathway analysis were performed in the standard enrichment computation method.
|
| |
|
| Submission date |
Sep 11, 2014 |
| Last update date |
Jan 01, 2015 |
| Contact name |
Anthony R Glover |
| E-mail(s) |
anthony.glover@sydney.edu.au
|
| Organization name |
University of Sydney
|
| Lab |
Cancer Genetics
|
| Street address |
Level 9, Kolling Institute of Medical Research, Royal North Shore Hospital
|
| City |
NSW |
| State/province |
St Leonards, Sydney |
| ZIP/Postal code |
2065 |
| Country |
Australia |
| |
|
| Platform ID |
GPL16956 |
| Series (1) |
| GSE61359 |
Long noncoding RNAs profiles of adrenocortical carcinomas, adrenocortical adenomas and normal adrenal cortex |
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