bloom richardson grade: 2 er: 1 pr: 1 her2: 0 ki67: 0 p53: 0 tissue: breast cancer tumor
Extracted molecule
genomic DNA
Extraction protocol
10 x 10 u slices containing at least 60% tumour cells were cleared of paraffin (2 x 5 min xylene, 2 x 30 s 100% ethanol, 30s 90% ethanol, 30 s 70% ethanol, and rinsed with H2O), treated with 1 M NaSCN at 37 C overnight, and sections of interest (>70% tumour cells) were scraped in 200 ul buffer ATL (Qiagen, cat. no. 51304). 27 ul of proteinase K (15 ug/ull, Roche, cat. no.3115879001) was immediately added, as well as at the end of the day, and at the beginning and end of the next day; samples were constantly shaken at 37 C during the time of digestion. The following day, 40 ul RNase A (20 ug/ul, Sigma, cat. no. R5500) was added to the sample, vortexed, and incubated for 2 min at room temperature. 400 ul of buffer AL (Qiagen, cat.no.51304) was added and incubated for 10 min at 70 C. 420 ul of 100% ethanol was added and vortexed. The sample mixture was spun on a spincolumn (Qiagen, cat. no. 51304) for 1 min at 8,000 rpm. The column was sequentially washed with 500 ul of the following reagents and spun for 1 min at 8,000 rpm: AW1, AW2, and twice with 80% ethanol. The column was spun dry for 3 min at 14,000 rpm. The sample was eluted with 50 ul of AE buffer by spinning for 1 min at 8,000 rpm. (Joosse, SA et al, Breast Cancer Res Treat, 2009)
Label
Cy3
Label protocol
With the ENZO 42760 labeling kit for array CGH: 20 uL primers (vial 1) with 500 ng of genomic tumor DNA or Promega Female reference DNA (GG1521) in 19 ul of nuclease free water for 10 minutes at 99 C in pcr machine, followed by 10 minutes of cooling to 4 degrees. Snap freeze and spin. While on ice add 11 ul Cy3 mix (10ul Cy3+1ul Klenow DNA pol (vial 4)) to tumor DNA, and 11 ul Cy5 mix (10ul Cy3+1ul Klenow DNA pol (vial 4)) to reference DNA and incubate in PCR machine for 4 hours at 37 C. Then add 5ul stop buffer (vial 5). To remove excess nucleotides with Qiagen MinElute PCR purification kit add 275 uL binding buffer (PB) to sample and reference. Put on minElute column and spin 60 sec on 16000 rcf. Add 700 ul Wash Buffer (PE) and centrifuge 60 secs at 16000 rcf. Dry columns by spinning another 60 secs at 16000 rcf. Elute the DNA in 2 steps by adding 2x 10.5 ul of elution buffer (EB). Pool equal amounts of tumor and reference DNA and speedvac into a pellet.
10 x 10 u slices containing at least 60% tumour cells were cleared of paraffin (2 x 5 min xylene, 2 x 30 s 100% ethanol, 30s 90% ethanol, 30 s 70% ethanol, and rinsed with H2O), treated with 1 M NaSCN at 37 C overnight, and sections of interest (>70% tumour cells) were scraped in 200 ul buffer ATL (Qiagen, cat. no. 51304). 27 ul of proteinase K (15 ug/ull, Roche, cat. no.3115879001) was immediately added, as well as at the end of the day, and at the beginning and end of the next day; samples were constantly shaken at 37 C during the time of digestion. The following day, 40 ul RNase A (20 ug/ul, Sigma, cat. no. R5500) was added to the sample, vortexed, and incubated for 2 min at room temperature. 400 ul of buffer AL (Qiagen, cat.no.51304) was added and incubated for 10 min at 70 C. 420 ul of 100% ethanol was added and vortexed. The sample mixture was spun on a spincolumn (Qiagen, cat. no. 51304) for 1 min at 8,000 rpm. The column was sequentially washed with 500 ul of the following reagents and spun for 1 min at 8,000 rpm: AW1, AW2, and twice with 80% ethanol. The column was spun dry for 3 min at 14,000 rpm. The sample was eluted with 50 ul of AE buffer by spinning for 1 min at 8,000 rpm. (Joosse, SA et al, Breast Cancer Res Treat, 2009)
Label
Cy5
Label protocol
With the ENZO 42760 labeling kit for array CGH: 20 uL primers (vial 1) with 500 ng of genomic tumor DNA or Promega Female reference DNA (GG1521) in 19 ul of nuclease free water for 10 minutes at 99 C in pcr machine, followed by 10 minutes of cooling to 4 degrees. Snap freeze and spin. While on ice add 11 ul Cy3 mix (10ul Cy3+1ul Klenow DNA pol (vial 4)) to tumor DNA, and 11 ul Cy5 mix (10ul Cy3+1ul Klenow DNA pol (vial 4)) to reference DNA and incubate in PCR machine for 4 hours at 37 C. Then add 5ul stop buffer (vial 5). To remove excess nucleotides with Qiagen MinElute PCR purification kit add 275 uL binding buffer (PB) to sample and reference. Put on minElute column and spin 60 sec on 16000 rcf. Add 700 ul Wash Buffer (PE) and centrifuge 60 secs at 16000 rcf. Dry columns by spinning another 60 secs at 16000 rcf. Elute the DNA in 2 steps by adding 2x 10.5 ul of elution buffer (EB). Pool equal amounts of tumor and reference DNA and speedvac into a pellet.
Hybridization protocol
According to manufacterer's protocol (Nimblegen Roche)
Scan protocol
2 um double pass
Data processing
Nimblescan, grid tiff files, generate pair reports followed by using the NimbleScan DNAcopy procedure at default settings
