|
| Status |
Public on Nov 08, 2016 |
| Title |
UCA8 |
| Sample type |
RNA |
| |
|
| Source name |
Colon tissue
|
| Organism |
Homo sapiens |
| Characteristics |
age: 26
gender: Female
location: colon
sample type: active ulcerative colitis
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using Trizol RNA extraction method
|
| 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 (Arraystar Flash RNA Labeling Kit, Arraystar). 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. 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.
|
| |
|
| Hybridization protocol |
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 v12.1 software package (Agilent Technologies). After quantile normalization of the raw data, LncRNAs and mRNAs that at least 19 out of 26 samples have flags in Present or Marginal (“All Targets Value”) were chosen for further data analysis. Differentially expressed LncRNAs and mRNAs were identified through Volcano Plot filtering between two groups. Hierarchical Clustering was performed using the R software. GO analysis and Pathway analysis were performed in the standard enrichment computation method
|
| |
|
| Submission date |
Jan 20, 2016 |
| Last update date |
Nov 08, 2016 |
| Contact name |
David Miguel Padua |
| E-mail(s) |
davepadua@gmail.com
|
| Organization name |
UCLA
|
| Department |
Medicine-Division of Digestive Diseases
|
| Lab |
.
|
| Street address |
10945 Le Conte Ave, PVUB 2114, MC 694907
|
| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90027 |
| Country |
USA |
| |
|
| Platform ID |
GPL16956 |
| Series (1) |
| GSE77013 |
A long noncoding RNA signature in Ulcerative Colitis |
|
