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Sample GSM865756 Query DataSets for GSM865756
Status Public on Sep 11, 2013
Title KASUMI-1, Ago4-associated 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: Ago4-associated miRNAs
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 Identification of Ago4-associated 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) pablo.landgraf@gmx.de
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

Data table header descriptions
ID_REF
VALUE Background corrected and normalized ratio (Channel2/Channel1) values.

Data table
ID_REF VALUE
1
2
3
4
5
6
7
8
9
10 1.766
11 0.291
12
13
14
15
16
17
18
19 0.272
20

Total number of rows: 1557

Table truncated, full table size 8 Kbytes.




Supplementary file Size Download File type/resource
GSM865756.gpr.gz 663.5 Kb (ftp)(http) GPR
Processed data included within Sample table

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