|
| Status |
Public on Feb 24, 2017 |
| Title |
EVI1 shRNA construct 2 3 |
| Sample type |
RNA |
| |
|
| Source name |
mammary gland/breast; derived from metastatic site: pleural effusion
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MDA-MB-231
cell type: breast carcinoma
|
| Treatment protocol |
Cells were lentivirally transduced as described in Konantz et al., Leukemia 2013
|
| Growth protocol |
The MDA-MB-231 cell line was cultivated according to the data sheet
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) and RNA concentration and purity determined on the NanoDrop ND-1000 spectral photometer (peqlab). The 2100 Bioanalyzer (Agilent Technologies) and the RNA 6000 Nano as well as the 6000 Pico LabChip Kit (Agilent Technologies) were used to analyze RNA integrity.
|
| Label |
Cy3
|
| Label protocol |
100 ng total RNA per sample were introduced into a reverse transcription with subsequent in vitro transcription (RT-IVT) reaction. Prior to RT-IVT, the total RNA samples were spiked with in vitro synthesized polyadenylated transcripts (One-Color RNA Spike-In Mix, Agilent Technologies) which serve as an internal labeling control for linearity, sensitivity and accuracy. The spiked total RNA was reverse transcribed into cDNA and then converted into Cyanine-3 labeled cRNA (Low Input Quick-Amp Labeling Kit One-Color, Agilent Technologies). All steps were carried out according to manufacturer’s instructions. cRNA concentration (ng/μl), RNA absorbance ratio (A260nm/A280nm) and Cyanine-3 dye concentration (pmol/μl) were recorded for all cRNA samples using NanoDrop ND-1000 UV- VIS spectrophotometer. Yield and specific activity of each reaction were determined. The RNA 6000 Nano LabChip Kit (Agilent Technologies) was used on the 2100 Bioanalyzer to analyze the quality of labeled non-fragmented cRNA.
|
| |
|
| Hybridization protocol |
Following cRNA clean-up and quantification 600 ng of each Cyanine-3-labeled cRNA sample was fragmented and prepared for one-color-based hybridization (Gene Expression Hybridization Kit, Agilent Technologies). Labeled cRNA samples were hybridized at 65°C for 17 hrs on separate Agilent SurePrint G3 Human Gene Expression 8x60K v2 Microarrays (AMADID 039494). Afterwards, microarrays were washed with increasing stringency using Gene Expression W ash Buffers (Agilent Technologies) followed by drying with acetonitrile (SIGMA).
|
| Scan protocol |
Fluorescent signal intensities were detected with Scan Control A.8.4.1 software (Agilent Technologies) on the Agilent DNA Microarray Scanner and extracted from the images using Feature Extraction 10.7.3.1 software (Agilent Technologies).
|
| Description |
BC9
|
| Data processing |
The software tools Feature Extraction 10.7.3.1, GeneSpring GX 12.6 (both Agilent Technologies), Excel 2010 (Microsoft) and the IMGM internal tool marfin v1.9 were used for quality control, statistical data analysis, filtering of differentially expressed transcript, RNA annotation and visualization.
|
| |
|
| Submission date |
Feb 23, 2017 |
| Last update date |
Feb 24, 2017 |
| Contact name |
Hui Wang |
| E-mail(s) |
hui.wang@unibas.ch
|
| Organization name |
University Hospital Basel
|
| Street address |
Hebelstr. 20
|
| City |
Basel |
| ZIP/Postal code |
4031 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL17077 |
| Series (1) |
| GSE95272 |
Prominent oncogenic roles of EVI1 in breast carcinoma |
|
