|
| Status |
Public on Jan 01, 2017 |
| Title |
FFPE DNA-Breast Cancer, Tissue Core 84 vs. Female Ref DNA |
| Sample type |
genomic |
| |
|
| Channel 1 |
| Source name |
Tissues specimen of primary FFPE breast cancer
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: clinical sample
tissue: tumor sample
diagnosis: breast cancer
gender: female
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA were isolated out of formalin-fixed, paraffin-embedded (FFPE) tissue specimens using the QIAamp DNA FFPE Tissue Kit (Qiagen) following the instructions published by the manufacturer (QIAamp DNA FFPE Tissue Handbook 06/2012). FFPE tissue specimens were excised from primary breast tumors using a sterile disposable biopsy punch (1.5mm diameter), excess paraffin was removed, residual tissue was manually minced using sterile scalpel blades before starting the DNA isolation. Following the DNA extraction DNA, DNA yield were quantified spectrophotometrically and the average size of extracted DNA molecules was assessed on 1.5% agarose gel supplemented with ethidium bromide. Reference DNA samples were extracted using the DNeasy blood and tissue Kit (Qiagen) from mononuclear cells obtained from peripheral blood following gradient centrifugation in 65% percoll soultion. DNA extraction was conducted following the protocol provided by the manufacturer (DNeasy Blood & Tissue Handbook 07/2006). High-molecular weight DNA in reference samples was sheared using Covaris S220x system down to approximately 500 bp (size matching the average fragments size of DNA in test samples) following guidelines provided by the manufacturer (microTUBE AFA fiber snap-cap 130µl tubes, peak incident power - 105W, duty factor - 5%, cycles per burst - 200, treatment time - 80s, Temperature 7 oC. Following the average DNA fragments size was assessed on 1.5% agarose gel supplemente with ethidium bromide.
|
| Label |
Cy5
|
| Label protocol |
DNA samples were labeled using Agilent SureTag DNA Labeling Kit (Cat #: 5190-3400) following instruction provided by the manufacturer (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis v7.3, March 2014). Slight modifications to the original protocol were introduced - i.e. restiriction digestion with AluI and RsaI was omitted.
|
| |
|
| Channel 2 |
| Source name |
Mononuclear cells enriched (gradient centrifugation in Percoll 65) from peripheral blood of a healthy donor_DNeasy Blood & Tissue Kit
|
| Organism |
Homo sapiens |
| Characteristics |
sample type: Clinical sample
tissue: peripheral blood
cell type: mononuclear cells
diagnosis: healthy donor
gender: female
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA were isolated out of formalin-fixed, paraffin-embedded (FFPE) tissue specimens using the QIAamp DNA FFPE Tissue Kit (Qiagen) following the instructions published by the manufacturer (QIAamp DNA FFPE Tissue Handbook 06/2012). FFPE tissue specimens were excised from primary breast tumors using a sterile disposable biopsy punch (1.5mm diameter), excess paraffin was removed, residual tissue was manually minced using sterile scalpel blades before starting the DNA isolation. Following the DNA extraction DNA, DNA yield were quantified spectrophotometrically and the average size of extracted DNA molecules was assessed on 1.5% agarose gel supplemented with ethidium bromide. Reference DNA samples were extracted using the DNeasy blood and tissue Kit (Qiagen) from mononuclear cells obtained from peripheral blood following gradient centrifugation in 65% percoll soultion. DNA extraction was conducted following the protocol provided by the manufacturer (DNeasy Blood & Tissue Handbook 07/2006). High-molecular weight DNA in reference samples was sheared using Covaris S220x system down to approximately 500 bp (size matching the average fragments size of DNA in test samples) following guidelines provided by the manufacturer (microTUBE AFA fiber snap-cap 130µl tubes, peak incident power - 105W, duty factor - 5%, cycles per burst - 200, treatment time - 80s, Temperature 7 oC. Following the average DNA fragments size was assessed on 1.5% agarose gel supplemente with ethidium bromide.
|
| Label |
Cy3
|
| Label protocol |
DNA samples were labeled using Agilent SureTag DNA Labeling Kit (Cat #: 5190-3400) following instruction provided by the manufacturer (Agilent Oligonucleotide Array-Based CGH for Genomic DNA Analysis v7.3, March 2014). Slight modifications to the original protocol were introduced - i.e. restiriction digestion with AluI and RsaI was omitted.
|
| |
|
| |
| 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 7.3, March 2014). 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 |
Reference DNA samples have been fragmented to an average size of 500bp (matching the average size of DNA samples extracted from FFPE specimens) with Covaris S220x system prior to labeling and hybridization
|
| 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. Threshold of the centralization algorithm was set to 6.0 and bin size of 10. To avoid false positive calls, aberration filters were applied to define the minimum log2 ratio (minimum average absolute log2 ratio = 0.25) and the minimum number of probes in an aberrant interval (minProbes = 25).
log2 ratio = Normalized log2 ratios (Cy5/Cy3) representing test/reference. The log2 ratios were obtained using the Agilent Genomic Workbench 6.5.0.18; Average log2 value of a genomic interval = average log2 ratios calculated for entire aberrant genomic intervals (0 indicates balance copy number state)
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| |
|
| Submission date |
Aug 31, 2016 |
| Last update date |
Jan 01, 2017 |
| 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) |
| GSE86305 |
HER2 FISH results in breast cancers with increased CEN17 signals using alternative chromosome 17 probes - reclassifying cases in the equivocal category. |
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