|
| Status |
Public on Sep 11, 2013 |
| Title |
KASUMI-1, Isotype control detected miRNAs, biological rep2 |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
synthetic miRNA pool (universal reference)
|
| Organism |
synthetic construct |
| Characteristics |
reference: 1 fmol of each of 771 synthetic miRNAs were pooled and labeled
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The 4 Ago antibodies were obtained from our cooperation partner Prof. Dr. Gunter Meister from University Regensburg and were manufactured in house. TRIzol extraction of Ago- / isotype control-associated and total RNA was performed according to the manufacturer's instructions.
|
| Label |
Cy3
|
| Label protocol |
miRNAs were 3' end labeled using a truncated and mutated RNA ligase Rnl2(1-249)K227Q
|
| |
|
| Channel 2 |
| Source name |
KASUMI-1 cell line, 4'-thiouridine and UV treated
|
| Organism |
Homo sapiens |
| Characteristics |
tissue: human acute myeloid leukemia
genotype: cells carry the t(8;21) AML1-ETO fusion gene
age: cells were harvested after 48 - 72 h
rna subtype: unspecific bound miRNAs of isotype control
|
| Extracted molecule |
total RNA |
| Extraction protocol |
The 4 Ago antibodies were obtained from our cooperation partner Prof. Dr. Gunter Meister from University Regensburg and were manufactured in house. TRIzol extraction of Ago- / isotype control-associated and total RNA was performed according to the manufacturer's instructions.
|
| Label |
Cy5
|
| Label protocol |
miRNAs were 3' end labeled using a truncated and mutated RNA ligase Rnl2(1-249)K227Q
|
| |
|
| |
| Hybridization protocol |
The corresponding Cy3 and Cy5 labled miRNAs were combined and hybridized overnight (16 hours, 42°C) to a miRXplore microarray (MACS molecular Miltenyi Biotec) using an a-Hyb Hybridization Station (MACS molecular Miltenyi Biotec). The microarrays were washed five times with distilled water (at room temperature) and dried with compressed, dry air.
|
| Scan protocol |
miRXplore microarrays were scanned using the Axon GenePix 4200A Microarray Scanner
|
| Description |
Isotype control detected miRNAs
|
| Data processing |
Microarray image analysis and ratio-based normalisation of the microarray data was conducted using the software package GenePix Pro 6.1 (Molecular Devices). Data was filtered with respect to background-subtracted signal intensity, signal to noise ratio and spot diameter. Local background was subtracted from the signal to obtain the net signal intensity and the ratio of both fluorescent labels. Subsequently, the mean of the ratios of 4 corresponding spots representing the same miRNA was computed for those spots only which were unflagged (empty spots, poor spots, negative spots) and for which the fluorescent intensity of the miRNAs derived from the samples of interest was two-fold the mean background value. The mean ratios of all probes were normalized to the median of the ratios detected for the spiked 18 synthetic RNA oligonucleotides reverse complement to miRControl 3 probes. Signal intensities of miRNAs without the corresponding miRNA in the synthetic miRNA pool were divided by the mean value of signal intensities of all detected miRNAs in the pool.
|
| |
|
| Submission date |
Jan 24, 2012 |
| Last update date |
Sep 11, 2013 |
| Contact name |
Pablo Landgraf |
| E-mail(s) |
[email protected]
|
| Organization name |
Heinrich-Heine University Düsseldorf
|
| Department |
Clinic of Pediatric Oncology, Hematology and Clinical Immunology
|
| Street address |
Moorenstraße 5
|
| City |
Düsseldorf |
| ZIP/Postal code |
40225 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10993 |
| Series (1) |
| GSE35320 |
microRNAs distinguish cytogenetic subgroups in pediatric AML and contribute to complex regulatory networks in AML-relevant pathways |
|
