|
| Status |
Public on Jan 13, 2014 |
| Title |
08-S-6658C |
| Sample type |
RNA |
| |
|
| Source name |
Control_FFPE tissue
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: FFPE tissue
sample type: Control
histology: Non-NPC tissue. Differentiated
|
| Treatment protocol |
Formalin Fixed Paraffin Embedded (FFPE) biopsy samples from four cases of histologically proven nasopharyngeal carcinoma.2 x 20 µm slices from each case were prepared for RNA purification.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from FFPE sections using the miRNeasy FFPE kit (Qiagen) according to manufacturer’s protocol. Briefly, 320 µL Deparaffinization Solution (Qiagen) was added followed by brief vortexing, centrifugation and incubation. Buffer PKD was added, samples centrifuged and proteinase K added. Digestion was performed at 56◦C for 15 minutes and 80◦C for 15 minutes in order to partially reverse formaldehyde modification. The lower phase was removed and DNase digestion performed at room temperature for 15 minutes. 500 µL RBC buffer was then added, followed by 100% Ethanol (Acros Chemical) and transferred to the RNeasy MiniElute column. The column was washed twice with RPE, followed by drying and then eluted with 30 µL RNase-free water
|
| Label |
Cy3
|
| Label protocol |
250ng Total RNA from each FFPE case was labeled with Cyanine 3-pCp by using Agilent miRNA labeling kits according to the manufacturer's instructions. Labeled samples were desalted by using MicroBioSpin 6 Columns (Bio-Rad).
|
| |
|
| Hybridization protocol |
Labeled total RNA samples were dried completely by using speed-vac, and then hybrized with probes on array slides, accorkding to the manufacture. The hybridization lasted for 20 hours inside 55'C hybridization oven (Agilent). After hybridization, arrays were washed with Gene Expression Wash Buffer 1 (Agilent) at room temperature, and then wahsed with Wash Buffer 2 (Agilent) at 37'C. The washed array were dried and processed to scanning step immediately.
|
| Scan protocol |
Slides was scanned immediated after washing step on Agilent G2565CA Microarray Scanner System. (Scan Area is 61x21.6 mm; Scan resolution is 3um; Color channel is Green; Green PMT is 100%)
|
| Data processing |
The feature intensities were transferred to digital data and Log2 transformed by using Feature Extraction (V.10.7) software on default setting. (Protocol is miRNA_107_Sep09, Grid is 031181_D_F_20111226). For data analysis, inter-sample variance was normalized by using quantile normalization strategies on GeneSpringGX 12.0.
|
| |
|
| Submission date |
Apr 18, 2013 |
| Last update date |
Jan 13, 2014 |
| Contact name |
Jeffrey Bethony |
| E-mail(s) |
jbethony@gwu.edu
|
| Organization name |
George Washington University
|
| Department |
Microbiology, Immunology, and Tropical Medicine
|
| Street address |
2300 Eye St. NW.
|
| City |
Washington |
| State/province |
DC |
| ZIP/Postal code |
20037 |
| Country |
USA |
| |
|
| Platform ID |
GPL16770 |
| Series (1) |
| GSE46172 |
Microarray analysis of microRNAs in NPC FFPE tissue samples |
|
