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| Status |
Public on Nov 05, 2014 |
| Title |
064N02_130925 |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Circulating tumor cells collected from peripheral blood of breast cancer patients
|
| Organism |
Homo sapiens |
| Characteristics |
patient id: 064
origin: Clinical Sample
breast cancer patient/heathy donor: breast cancer patient
gender: female
enrichemnt/isolation procedure: CellSearch® System/DEPArray™ System
cell type: White Blood Cell (WBC)
expressed markers: Cytokeratin (+); CD45(+); DAPI(+)
product: Single-cell WGA product
method of whole genome amplification (wga): Ampli1™ WGA Kit (Silicon Biosystems)
patient id: Reference Female
enrichemnt/isolation procedure: Percoll density centrifugation/Manual isolation of single cells
|
| Treatment protocol |
Cancer patients: 7.5 mL of peripheral blood was collected into a CellSave preservative tube. Following enrichment and enumeration of circulating tumor cells (CTCs) by use of the CellSearch System, single CTCs, single white blood cells (WBCs) and CTC cell pools were collected using the DEPArray System. Single-cell specimens and cell pools were subjected to Ampli1 WGA protocol. The quality of the resulting WGA products was assessed using the Ampli1 QC Kit. Healthy donor: 10 mL of peripheral blood was collected from a healthy female donor. Mononuclear cell fraction was enriched using centrifugation in density gradient of Percoll. Single-cell were picked in a manual fashion using the micromanipulator and subjected to whole genome amplification using the Ampli1 WGA Kit. The quality of the resulting WGA products was assessed using the Ampli1 QC Kit. Four high quality single-cell WGA products were pooled and used as reference sample for array CGH.
|
| Growth protocol |
Not applicable. Samples for the analysis were collected from peripheral blood. No cell culture protocols were applied on the blood cells prior to sample processing.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cancer patients: following the enrichment with the CellSearch System single cells and cell pools were extracted using the DEPArray System. Subsequently, DNA from single-cell and cell pools were amplified using the Ampli1 WGA Kit. Healthy donors: single-cell were collected in non-automatized manner using an inverted microscope equipped with a micromanipulator. Single-cell specimens were processed using the Ampli1 WGA Kit.
|
| Label |
Cy5
|
| Label protocol |
Single-cell WGA samples were labeled using a previously published PCR-based approach. Prior to labeling, the primary WGA products were reamplified by PCR. Reamplification of the WGA products and the subsequent labeling were conducted as decribed by Czyz ZT et al., PLoS One. 2014 Jan 21;9(1):e85907.
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| |
|
| Channel 2 |
| Source name |
White blood cells (WBCs) collected from peripheral blood of ahealthy female individual
|
| Organism |
Homo sapiens |
| Characteristics |
origin: Clinical Sample
breast cancer patient/heathy donor: healthy donor
gender: female
cell type: White Blood Cell (WBC)
product: Mix of 4 single-cell WGA products
method of whole genome amplification (wga): Ampli1™ WGA Kit (Silicon Biosystems)
|
| Treatment protocol |
Cancer patients: 7.5 mL of peripheral blood was collected into a CellSave preservative tube. Following enrichment and enumeration of circulating tumor cells (CTCs) by use of the CellSearch System, single CTCs, single white blood cells (WBCs) and CTC cell pools were collected using the DEPArray System. Single-cell specimens and cell pools were subjected to Ampli1 WGA protocol. The quality of the resulting WGA products was assessed using the Ampli1 QC Kit. Healthy donor: 10 mL of peripheral blood was collected from a healthy female donor. Mononuclear cell fraction was enriched using centrifugation in density gradient of Percoll. Single-cell were picked in a manual fashion using the micromanipulator and subjected to whole genome amplification using the Ampli1 WGA Kit. The quality of the resulting WGA products was assessed using the Ampli1 QC Kit. Four high quality single-cell WGA products were pooled and used as reference sample for array CGH.
|
| Growth protocol |
Not applicable. Samples for the analysis were collected from peripheral blood. No cell culture protocols were applied on the blood cells prior to sample processing.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cancer patients: following the enrichment with the CellSearch System single cells and cell pools were extracted using the DEPArray System. Subsequently, DNA from single-cell and cell pools were amplified using the Ampli1 WGA Kit. Healthy donors: single-cell were collected in non-automatized manner using an inverted microscope equipped with a micromanipulator. Single-cell specimens were processed using the Ampli1 WGA Kit.
|
| Label |
Cy3
|
| Label protocol |
Single-cell WGA samples were labeled using a previously published PCR-based approach. Prior to labeling, the primary WGA products were reamplified by PCR. Reamplification of the WGA products and the subsequent labeling were conducted as decribed by Czyz ZT et al., PLoS One. 2014 Jan 21;9(1):e85907.
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| |
|
| |
| 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) with slight modifications. 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 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 and 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.
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| 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 |
Genomic profiling of single white blood cells (WBCs). WBCs were used as control samples to validate the assay and to select the most optimal settings to process single-cells extracted from the CellSearch System
|
| 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 6.5.0.18 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 aberration calls, aberration filters were applied to define the minimum log2 ratio (minAvgAbsLogRatio = 0.25) and the minimum number of probes in an aberrant interval (minimum number of probes in an interval = 50).
The normalization algorithm is an integral part of the standard data extraction/normalization algorithm (CGH_107_Sep09) protocol implemented in the Agilent Feature Extraction Software v10.7. Exactly this version of software in combination with extraction/normalization algorithm stated above were used to process the data generated for my submission.
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| |
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| Submission date |
Jun 03, 2014 |
| Last update date |
Nov 06, 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) |
| GSE58192 |
Molecular profiling of single circulating tumor cells with diagnostic intention |
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