|
| Status |
Public on Sep 28, 2011 |
| Title |
Cancer 4 |
| Sample type |
RNA |
| |
|
| Source name |
Cancer cells, pancreas,xeograft, fresh frozen, microdissected
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Cancer cells, pancreas,xeograft, fresh frozen, microdissected
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation from microdissected cells was performed using the RNAqueous-Micro kit (Ambion); RNA from macrodisected cryostat sections was isolated using the mirVana PARIS RNA Isolation Kit (Ambion). In all instances RNA isolation was performed according to the manufacturer’s instructions. Total-RNA concentration and purity of total-RNA samples was measured with the NanoDrop 1000 spectrophotometer (NanoDrop Technologies/Thermo Scientific). Furthermore, total-RNA used for microarray analyses were further quantified using the Qubit RNA Assay kit (Invitorgen)
|
| Label |
Cy3
|
| Label protocol |
miRNA labeling was performed as described in Agilent miRNA microarray protocol version 2.0 using Agilent miRNA labeling kit. 100 ng of total RNA was dephosphorylated with calf intestine alkaline phosphatase for 30 min at 37°C. Denaturation was performed by adding DMSO and incubating at 100°C for 6 min and immediately transferred to ice water bath. Ligation was performed with pCp-Cy3 at 16 °C for 2 h. The labeled samples were dried completely in a vaccum concentrator and resuspended in 18 ul of nuclease free water.
|
| |
|
| Hybridization protocol |
The hybridization mixture [10X GE blocking agent (4.5 ul), 2X Hi-RPM hybridization buffer (22.5ul)] along with labeled miRNA sample was heated for 5 min at 100 °C and immediately cooled to 0 °C. Each 45 mL sample was hybridized onto a microarray at 55 °C for 21 h. Slides were washed 5 min in GE wash buffer 1 at RT and again for 5 min in GE wash buffer 2 at 37 °C, followed by an acetonitrile wash for 10 sec at RT to dry the slides completely.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent DNA Microarray Scanner (G2505B ) using one color scan setting for 8x15k array slides (Scan Area 61x21.6 mm, Scan resolution 5um, Dye channel is set to Green and Green PMT is set to 100%, No XDR).
|
| Description |
miRNA expression
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol miRNA_107_Sep09 and Grid: 019118_D_F_20100604) to obtain background subtracted and spatially detrended Processed Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
|
| |
|
| Submission date |
Dec 02, 2010 |
| Last update date |
Sep 28, 2011 |
| Contact name |
Stephan A Hahn |
| E-mail(s) |
stephan.hahn@rub.de
|
| Phone |
0049 234 3229282
|
| Organization name |
Ruhr-University Bochum
|
| Department |
MGO
|
| Street address |
Universitaetsstrasse 150
|
| City |
Bochum |
| ZIP/Postal code |
44892 |
| Country |
Germany |
| |
|
| Platform ID |
GPL7731 |
| Series (1) |
| GSE25820 |
Global microRNA expression profiling of microdissected tissues from diseased and non-diseased pancreatic tissues |
|
