|
| Status |
Public on Dec 01, 2014 |
| Title |
Normal adjacent breast tissue [67] |
| Sample type |
RNA |
| |
|
| Source name |
Breast tissue, fresh frozen
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: Breast
gender: Female
age: 67
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from human breast cancers and their matched adjacent normal tissues using the TRIzol® Plus RNA Purification Kit (Life Technologies, Grand Island NY) following the manufacturer's recommendations. RNA quantity and quality were measured by NanoDrop ND-2000 and RNA integrity was assessed by standard denaturing agarose gel electrophoresis.
|
| Label |
Cy3
|
| Label protocol |
Sample labeling was performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technology) with minor modifications. Briefly, mRNA was purified from total RNA after removal of rRNA (mRNA-ONLY™ Eukaryotic mRNA Isolation Kit, Epicentre). 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).
|
| Description |
LncRNA expression in normal adjacent breast tissue of patient 1
|
| 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 2 out of 4 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis. Differentially expressed LncRNAs and mRNAs were identified through Fold Change filtering between two samples. 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 |
Aug 22, 2014 |
| Last update date |
Dec 01, 2014 |
| Contact name |
Liuqing Yang |
| E-mail(s) |
lyang7@mdanderson.org
|
| Phone |
713-563-2654
|
| Organization name |
The University of Texas MD Anderson Cancer Center
|
| Department |
Molecular and Cellular Oncology
|
| Street address |
1515 Holcombe Blvd.
|
| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
| |
|
| Platform ID |
GPL16956 |
| Series (1) |
| GSE60689 |
Identification of LncRNA Expression Signatures for Triple-negative Breast Cancer |
|
