|
| Status |
Public on Oct 20, 2014 |
| Title |
PBL #1 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Peripheral blood leukocyte of healthy volunteer
|
| Organism |
Homo sapiens |
| Characteristics |
cell line/clinical sample: clinical sample
tumor sample/healthy donor: healthy donor
amplified/unamplified dna: amplified - Ampli1 WGA
primary/reamplified wga: reamplified WGA (Czyz et al., 2014, PLoS One)
labeling protocol: PCR-based labeling (Czyz et al., 2014, PLoS One)
gender: male
primary/reamplified wga: reamplified WGA (Czyz et al., 2014, PLoS One)
|
| Growth protocol |
VCaP cell line was grown in Dulbecco's Modified Eagle's Medium (PAN-Biotech) supplemented with 10% fetal calf serum (FCS) (PAN-Biotech), 4 mM L-Glutamic acid (PAN-Biotech) and 1% of Penicillin/Streptomycin solution (PEN STREP) (PAN-Biotech).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total genomic DNA (bulk DNA) was extracted using the Dbeasy Blood & Tissue Kit (QIAgen) following manufacture's instructions. All single cells, cell pools (few hundred cells) and microdissected specimens from formalin fixed paraffin embedded (FFPE) tumor tissue were amplified using the Ampli1™ WGA Kit (Silicon Biosystems).
|
| Label |
Cy5
|
| Label protocol |
PCR-based labeling using incorporation of dye-conjugated dNTPs. WGA products were labeled by PCR in the presence of Cy5 or Cy3 conjugated dCTP and dUTP. Incorporation of the fluorescent dyes using the dNTPs provided the advantage that adapters flanking each amplicon in the WGA product could be removed prior to aCGH hybridization, thereby avoiding unspecific cross-hybridization of test sample with reference DNA and oligonucleotide probes on the array. For each sample two 50µl PCR reactions were run in parallel each comprising 5 µl of 10x Expanded Long Template Buffer 1 (Roche Diagnostics), 2.4 µM of the universal primer (LIB1 or MseLig-21, depending on the adapter sequence incorporated in the WGA product), 350 µM of dATP and dGTP, 315 µM of dCTP and dTTP, 35 µM of Cy5/Cy3-dCTP and Cy5/Cy3-dUTP (GE Healthcare), 0.5 µl of BSA (Roche Diagnostics), 7.5 U of Expand-Long-Template DNA Polymerase (Roche) and 0.5 µl of the template DNA. Labeling products were pooled and purified using the Amicon Ultra 0.5 System (100 kDa cut-off). DNA yields and dye incorporation rates were quantified using the NanoDrop ND-1000 Instrument.
|
| |
|
| Channel 2 |
| Source name |
Healthy donor: cord blood; a pool of 4 single-cell WGA products from the same donor
|
| Organism |
Homo sapiens |
| Characteristics |
cell line/clinical sample: Clinical sample
tumor sample/healthy donor: healthy donor
amplified/unamplified dna: amplified - Ampli1 WGA
labeling protocol: PCR-based labeling (Czyz et al., 2014, PLoS One)
gender: male
|
| Growth protocol |
VCaP cell line was grown in Dulbecco's Modified Eagle's Medium (PAN-Biotech) supplemented with 10% fetal calf serum (FCS) (PAN-Biotech), 4 mM L-Glutamic acid (PAN-Biotech) and 1% of Penicillin/Streptomycin solution (PEN STREP) (PAN-Biotech).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total genomic DNA (bulk DNA) was extracted using the Dbeasy Blood & Tissue Kit (QIAgen) following manufacture's instructions. All single cells, cell pools (few hundred cells) and microdissected specimens from formalin fixed paraffin embedded (FFPE) tumor tissue were amplified using the Ampli1™ WGA Kit (Silicon Biosystems).
|
| Label |
Cy3
|
| Label protocol |
PCR-based labeling using incorporation of dye-conjugated dNTPs. WGA products were labeled by PCR in the presence of Cy5 or Cy3 conjugated dCTP and dUTP. Incorporation of the fluorescent dyes using the dNTPs provided the advantage that adapters flanking each amplicon in the WGA product could be removed prior to aCGH hybridization, thereby avoiding unspecific cross-hybridization of test sample with reference DNA and oligonucleotide probes on the array. For each sample two 50µl PCR reactions were run in parallel each comprising 5 µl of 10x Expanded Long Template Buffer 1 (Roche Diagnostics), 2.4 µM of the universal primer (LIB1 or MseLig-21, depending on the adapter sequence incorporated in the WGA product), 350 µM of dATP and dGTP, 315 µM of dCTP and dTTP, 35 µM of Cy5/Cy3-dCTP and Cy5/Cy3-dUTP (GE Healthcare), 0.5 µl of BSA (Roche Diagnostics), 7.5 U of Expand-Long-Template DNA Polymerase (Roche) and 0.5 µl of the template DNA. Labeling products were pooled and purified using the Amicon Ultra 0.5 System (100 kDa cut-off). DNA yields and dye incorporation rates were quantified using the NanoDrop ND-1000 Instrument.
|
| |
|
| |
| Hybridization protocol |
Array CGH was performed on oligonucleotide-based SurePrint G3 Human CGH 4x180K microarray slides (design code: 022060) according the protocol provided by the manufacturer (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis, version 7.1, December 2011). Slight modifications were introduced WGA products processed with the PCR-based labeling approaches. Here, the hybridization mix consisted of 5.0 µg of Cot1-DNA (Roche Diagnostics), 12 µl of 10x Blocking Reagent (Agilent Technologies), 60 µl of 2x Hi RPM Hybridization Buffer, 1% (v/v) of both Tween20 and Igepal and 19 µl of both test and reference DNA. For each hybridization 100 µl of the hybridization mix was applied on the array and hybridized at 65◦C for 24 h. Following the hybridization, the slides were washed sequentially: twice for 2:30 min in Oligo aCGH/ChIP-on-Chip Wash Buffer 1 (Agilent Technologies) at room temperature, twice for 30 sec min in Oligo aCGH/ChIP-on-Chip Wash Buffer 2 (Agilent Technologies) at 37◦C. Washed slides were immersed in acetonitrile to remove all remaining traces of the wash buffers.
|
| Scan protocol |
Scanned on an Agilent G2565CA scanner (Dye channel: R+G; Scan resolution: 2.5µm, double pass; Tiff file dynamic range: 16 Bit; Red PMT gain: 100%; Green PMT gain: 100%; No XDR)
|
| Description |
Sample used to demonstrate the performance of single cell aCGH - negative control. Data shown in Supplementary Figure of the manuscript.
|
| Data processing |
Microarray TIFF image files were processed with the Agilent Genomic Feature Extraction Software (version 10.7.3.1). The resulting text files were imported and analyzed with Agilent Genomic Workbench Software (version 7.0 Lite). Aberrant regions were recognized using ADM-2 algorithm with threshold set to 7.0. Centralization algorithm was set to a threshold of 6.0 and bin size of 10. To avoid false positive calls, aberration filters were applied to define the minimum log2 ratio (0.4 ) and the minimum region size of 10000 kbp.
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| |
|
| Submission date |
Jul 21, 2014 |
| Last update date |
Oct 20, 2014 |
| Contact name |
Zbigniew Tadeusz Czyz |
| E-mail(s) |
zbigniew.czyz@klinik.uni-regensburg.de
|
| Organization name |
Fraunhofer Institute for Toxicology and Experimental Medicine
|
| Department |
Project Group Personalized Tumor Therapy, Regensburg
|
| Street address |
Josef-Engert-Strasse 9
|
| City |
Regensburg |
| ZIP/Postal code |
93053 |
| Country |
Germany |
| |
|
| Platform ID |
GPL10150 |
| Series (1) |
| GSE59631 |
Microarray correpsonding to the paper manuscript titled: Targeted expression profiling of single disseminated cancer cells isolated from bone marrow of prostate cancer patients |
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