|
| Status |
Public on Mar 31, 2010 |
| Title |
Acute myeloid leukemia (AML-M3), first diagnosis, before treatment L77 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
acute leukemia
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
age: 43
sample: human acute myeloid leukemia (AML-M3), first diagnosis, before treatment
protocol: MCIp enrichment
disease state: AML-M3
|
| Growth protocol |
Leukemic blasts and bone marrow cells from AML patients were collected during routine diagnostic bone marrow aspirations. Patients had given informed consent to additional sample collection and analyses according to a protocol approved by the local ethical committee. DNA was prepared after Ficoll gradient centrifugation using Qiagen Blood & Cell Culture DNA Kit. DNA concentration was determined with the NanoDrop spectrophotometer and quality was assessed by agarose gel electrophoresis.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MCIp enrichment of methylated DNA was performed essentially as described (Gebhard et al. 2006, Cancer Research). In brief, genomic DNA was sonicated to a mean fragment size of 350-400 bp. Two µg of each sample were incubated with 150 µl Protein A-Sepharose 4 Fast Flow beads (GE Healthcare) coated with 60 µg purified MBD-Fc protein in 2 ml Ultrafree-MC centrifugal filter devices (Amicon/Millipore) for 3 h at 4°C in buffer containing 300 mM NaCl. Beads were centrifuged to recover unbound DNA fragments (300 mM fraction) and subsequently washed with buffers containing increasing NaCl concentrations (400, 500, 550 mM). Densely CpG-methylated DNA was eluted with a high-salt buffer (1000 mM NaCl) and all fractions were desalted using the MinElute PCR purification kit (Qiagen). The separation of CpG methylation densities of individual MCIp fractions was controlled by qPCR using primers covering the imprinted SNRPN
|
| Label |
Alexa Fluor 5–dCTP
|
| Label protocol |
Enriched methylated DNA fragments of the high-salt MCIp fractions were labeled with Alexa Fluor 5–dCTP (cancer cells) and Alexa Fluor 3–dCTP (normal cells) using the BioPrime Total Genomic Labeling System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
|
| |
|
| Channel 2 |
| Source name |
peripheral blood monocytes
|
| Organism |
Homo sapiens |
| Characteristics |
gender: male
cell type: peripheral blood monocytes
sample: mixture of three young (age 20-30) and healthy donors
protocol: MCIp enrichment
disease state: normal
|
| Growth protocol |
Leukemic blasts and bone marrow cells from AML patients were collected during routine diagnostic bone marrow aspirations. Patients had given informed consent to additional sample collection and analyses according to a protocol approved by the local ethical committee. DNA was prepared after Ficoll gradient centrifugation using Qiagen Blood & Cell Culture DNA Kit. DNA concentration was determined with the NanoDrop spectrophotometer and quality was assessed by agarose gel electrophoresis.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MCIp enrichment of methylated DNA was performed essentially as described (Gebhard et al. 2006, Cancer Research). In brief, genomic DNA was sonicated to a mean fragment size of 350-400 bp. Two µg of each sample were incubated with 150 µl Protein A-Sepharose 4 Fast Flow beads (GE Healthcare) coated with 60 µg purified MBD-Fc protein in 2 ml Ultrafree-MC centrifugal filter devices (Amicon/Millipore) for 3 h at 4°C in buffer containing 300 mM NaCl. Beads were centrifuged to recover unbound DNA fragments (300 mM fraction) and subsequently washed with buffers containing increasing NaCl concentrations (400, 500, 550 mM). Densely CpG-methylated DNA was eluted with a high-salt buffer (1000 mM NaCl) and all fractions were desalted using the MinElute PCR purification kit (Qiagen). The separation of CpG methylation densities of individual MCIp fractions was controlled by qPCR using primers covering the imprinted SNRPN
|
| Label |
Alexa Fluor 3–dCTP
|
| Label protocol |
Enriched methylated DNA fragments of the high-salt MCIp fractions were labeled with Alexa Fluor 5–dCTP (cancer cells) and Alexa Fluor 3–dCTP (normal cells) using the BioPrime Total Genomic Labeling System (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions.
|
| |
|
| |
| Hybridization protocol |
Comparative MCIp hybridizations on CpG island oligonucleotide microarrays (Agilent) were performed using the recommended, stringent protocol (Agilent).
|
| Scan protocol |
Scanned on an Agilent scanner at 5 µm resolution.
Images were analysed and data extracted using Agilent Feature Extraction Software (version 9.5.1).
|
| Description |
n/a
|
| Data processing |
Linear normalized, background subtracted VALUE data were obtained from log10 transformed, processed Red signal/processed Green signal values derived from image analysis with Agilent Feature Extraction Software (version 9.5.1). Processed signal intensities were further normalized using GC-dependent regression and imported into Microsoft Office Excel 2007 for further analysis.
|
| |
|
| Submission date |
Aug 05, 2009 |
| Last update date |
Mar 31, 2010 |
| Contact name |
Michael Rehli |
| E-mail(s) |
michael.rehli@klinik.uni-r.de
|
| Organization name |
University Hospital Regensburg
|
| Department |
Internal Med III
|
| Street address |
F.-J.-Strauss-Allee 11
|
| City |
Regensburg |
| ZIP/Postal code |
93042 |
| Country |
Germany |
| |
|
| Platform ID |
GPL8544 |
| Series (1) |
| GSE17510 |
Detection of aberrant DNA methylation in acute leukemia samples compared to normal human monocytes |
|
