|
| Status |
Public on Jul 01, 2013 |
| Title |
Q590_Tumour_Roche_replicate 3 |
| Sample type |
genomic |
| |
|
| Source name |
Breast Tumour
|
| Organism |
Homo sapiens |
| Characteristics |
year of diagnosis: 2010
sample type: Surgical
tissue type: FFPE
tissue histology: IDC-NST, grade 3
tumor immunophenotype: ER +ve; PgR +ve; HER2 3+
dna extraction method: Roche
sodium thiocyanate treatment performed: YES
ffpe restore performed: YES
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Tumour cores were punched from the FFPE blocks to enrich for tumour epithelial cells. Extraction of genomic DNA from these FFPE cores was performed using two different methods: the DNeasy Blood and Tissue Kit (QIAGEN) and the High Pure DNA Template Preparation Kit (Roche). Both methods were performed according to the manufacturer’s instructions with the following exceptions for both techniques: (i) some tissue samples were pre-treated in 1M sodium thiocyanate overnight at 37oC to remove cross-links and (ii) for all cases an extended tissue digestion step was performed over three nights with supplementary Proteinase K added every 24 hours. Eluted DNA was assessed for purity using the Nanodrop-2000 and double stranded DNA was quantified using the Qubit® fluorometer (Invitrogen) as per manufacturer’s instructions.
|
| Label |
C-Bio and A-DNP
|
| Label protocol |
100ng of DNA was subjected to FFPE DNA Restore. DNA was labelled according to the Infiniium HD FFPE Assay protocol. The genomic DNA samples were denatured using 0.1N NaOH, neutralised using RPM buffer before being whole-genome amplified in an overnight reaction at 37C using the MSM amplification master mix. After incubation the amplified DNA was fragmented with a fragmentation mix (FMS), precipitated with isopropanol and a precipitation mix (PM1) and resuspended in hybridization buffer (RA1).
|
| |
|
| Hybridization protocol |
RA1 resuspended DNA was loaded onto BeadChips arrays and incubated overnight at 48C. After hybridisation, BeadChips were washed and subjected to single-base extension and allele-specific staining.
|
| Scan protocol |
BeadChips were coated with XC4/ethanol , dried for 1 hour and scanned on an iScan System (Illumina) using 'Infinium HD' scan settings.
|
| Description |
5995908006_R06C01
|
| Data processing |
Overall SNP call rates and B allele frequency (BAF) and log R ratio (LRR) values for each SNP were extracted, calculated and exported using GenomeStudio version2010.3 (Illumina). The BAF, LRR and copy number calls were visualized/calculated using the genoCN method (Sun et al. Nucleic Acids Research 2009; 37(16): 5365-5377), implemented in R version 2.15.0.
|
| |
|
| Submission date |
Jan 10, 2013 |
| Last update date |
Jul 01, 2013 |
| Contact name |
Peter Simpson |
| E-mail(s) |
[email protected]
|
| Organization name |
The University of Queensland
|
| Department |
UQ Centre for Clinical Research
|
| Street address |
Royal Brisbane & Women's Hospital
|
| City |
Brisbane |
| ZIP/Postal code |
QLD 4029 |
| Country |
Australia |
| |
|
| Platform ID |
GPL16483 |
| Series (1) |
| GSE43406 |
Evaluating the restoration of DNA derived from archival formalin-fixed paraffin embedded tissues for genomic profiling by SNP-CGH analysis |
|
