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Subject Item: _:vb2203435
rdf:type: n9:Section
dc:title: methods
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Subject Item: _:vb2203436
rdf:type: n2:Context
rdf:value: DNA was isolated from the breast cancer cell-line SKBR3 (obtained from ATCC) or from FFPE tumor tissue with at least 70% tumor cells as described before [>>6<<]. Two micrograms of total genomic DNA were labeled with ULS-Cy5 according to the manufacturers' instructions (Kreatech Biotechnology, Amsterdam).
n2:mentions: n3:16333309

Subject Item: _:vb2203437
rdf:type: n2:Context
rdf:value: Corning CodeLink® slides containing the human 3.5 k BAC/PAC genomic clone set in triplicate were used as before [>>6<<]. As optimization target, we used CGH profiles of 6 FFPE tumors containing at least 70% tumor cells and the SKBR3 cell-line profile, obtained by the manual hybridization method described before [6]. Automated hybridizations were done in
n2:mentions: n3:16333309

Subject Item: _:vb2203438
rdf:type: n2:Context
rdf:value: As optimization target, we used CGH profiles of 6 FFPE tumors containing at least 70% tumor cells and the SKBR3 cell-line profile, obtained by the manual hybridization method described before [>>6<<]. Automated hybridizations were done in 63.5 × 21 mm chambers in a Tecan HS4800 Pro™ hybridization station, which uses liquid agitation during hybridization. Experiments involving human tissues were conducted with permission of our
n2:mentions: n3:16333309

Subject Item: _:vb2203439
rdf:type: n2:Context
rdf:value: Data processing included signal intensity measurement in ImaGene Software followed by median pintip (c.q. subarray) normalization and plotting in custom Matlab code as before [>>6<<].
n2:mentions: n3:16333309

Subject Item: _:vb2203440
rdf:type: n2:Context
rdf:value: For each CGH profile, we calculated the variance across all log2 ratios relative to the ratios of the underlying true ploidy levels as estimated by CGH-segmentation [>>16<<], secondly, we defined the dynamic range as the difference between the minimum log2 ratio and the maximum log2 ratio calculated by CGH-segmentation [16], and the average of all the standard deviations of the triplicate spot measurements
n2:mentions: n3:15705208

Subject Item: _:vb2203441
rdf:type: n2:Context
rdf:value: to the ratios of the underlying true ploidy levels as estimated by CGH-segmentation [16], secondly, we defined the dynamic range as the difference between the minimum log2 ratio and the maximum log2 ratio calculated by CGH-segmentation [>>16<<], and the average of all the standard deviations of the triplicate spot measurements of each probe was used as a third statistic.
n2:mentions: n3:15705208

Subject Item: _:vb2203442
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dc:title: background
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Subject Item: _:vb2203443
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rdf:value: Array CGH has become a successful and valuable tool for the analysis of chromosome copy-number alterations including the detection of sub-megabase alterations and has been applied to e.g. cell-lines, (tumor) tissues, and lymphocytes [>>1<<-5]. The power of aCGH technology to detect low-level copy number changes is critically dependent on DNA quality (e.g. DNA fragmentation and cross-links) and sample heterogeneity.
n2:mentions: n3:9541108 n3:16417655 n3:1359641 n3:12297621

Subject Item: _:vb2203444
rdf:type: n2:Context
rdf:value: Therefore, selection of DNA of sufficient quality, especially when using FFPE material, is of great importance for aCGH [>>6<<]. Furthermore, whole genome amplification may be performed when insufficient DNA is available from a sample [7-9]. In addition to sample quality, enzymatic labeling protocols decrease average DNA size further which results in increased
n2:mentions: n3:16333309

Subject Item: _:vb2203445
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rdf:value: Furthermore, whole genome amplification may be performed when insufficient DNA is available from a sample [>>7<<-9]. In addition to sample quality, enzymatic labeling protocols decrease average DNA size further which results in increased noise due to non-specific binding [10], especially when the average PCR length of the sample template drops below
n2:mentions: n3:16271290 n3:16751780 n3:15681476

Subject Item: _:vb2203446
rdf:type: n2:Context
rdf:value: In addition to sample quality, enzymatic labeling protocols decrease average DNA size further which results in increased noise due to non-specific binding [>>10<<], especially when the average PCR length of the sample template drops below 200 bp [6].
n2:mentions: n3:10379877

Subject Item: _:vb2203447
rdf:type: n2:Context
rdf:value: to sample quality, enzymatic labeling protocols decrease average DNA size further which results in increased noise due to non-specific binding [10], especially when the average PCR length of the sample template drops below 200 bp [>>6<<]. As an alternative, chemical labeling protocols with cyanin cis-platinum-labeled DNA resulted in good aCGH results [11], also for FFPE archival samples [6].
n2:mentions: n3:16333309

Subject Item: _:vb2203448
rdf:type: n2:Context
rdf:value: As an alternative, chemical labeling protocols with cyanin cis-platinum-labeled DNA resulted in good aCGH results [>>11<<], also for FFPE archival samples [6].
n2:mentions: n3:15283211

Subject Item: _:vb2203449
rdf:type: n2:Context
rdf:value: As an alternative, chemical labeling protocols with cyanin cis-platinum-labeled DNA resulted in good aCGH results [11], also for FFPE archival samples [>>6<<]. One of the challenges of aCGH is its lower hybridization signal-intensity compared with metaphase-CGH. Based on literature and our previous experiments we hypothesized that the hybridization improves with increasing effective
n2:mentions: n3:16333309

Subject Item: _:vb2203450
rdf:type: n2:Context
rdf:value: There have been earlier reports on array CGH of FFPE material [>>6<<-8,12-14] and other reports on automated hybridization [15].
n2:mentions: n3:16751780 n3:16271290 n3:16333309

Subject Item: _:vb2203451
rdf:type: n2:Context
rdf:value: There have been earlier reports on array CGH of FFPE material [6-8,>>12<<-14] and other reports on automated hybridization [15].
n2:mentions: n3:15957152 n3:16467165 n3:16446704

Subject Item: _:vb2203452
rdf:type: n2:Context
rdf:value: There have been earlier reports on array CGH of FFPE material [6-8,12-14] and other reports on automated hybridization [>>15<<]. This report however, is the first that combines automated hybridization of FFPE tumor material on a BAC array, using non-enzymatic labeling and provides a method without formamide in the post-hybridization washes.
n2:mentions: n3:16175506

Subject Item: _:vb2203453
rdf:type: n9:Section
dc:title: results and discussion
n9:contains: _:vb2203454 _:vb2203455 _:vb2203456 _:vb2203457 _:vb2203458 _:vb2203459 _:vb2203460 _:vb2203461 _:vb2203462 _:vb2203463 _:vb2203464 _:vb2203465 _:vb2203466 _:vb2203467

Subject Item: _:vb2203454
rdf:type: n2:Context
rdf:value: Therefore, we chose to use the widely studied cell-line SKBR3 as a model for which chromosomal aberrations have been well documented [>>2<<,3,17], although the existence of minor sub-clone related alterations cannot be ruled out.
n2:mentions: n3:12297621

Subject Item: _:vb2203455
rdf:type: n2:Context
rdf:value: Therefore, we chose to use the widely studied cell-line SKBR3 as a model for which chromosomal aberrations have been well documented [2,>>3<<,17], although the existence of minor sub-clone related alterations cannot be ruled out.
n2:mentions: n3:16417655

Subject Item: _:vb2203456
rdf:type: n2:Context
rdf:value: In a previous study [>>6<<], we performed manual hybridizations of over one hundred BAC arrays that helped us to develop the quality criteria that were now used to optimize automated hybridization.
n2:mentions: n3:16333309

Subject Item: _:vb2203457
rdf:type: n2:Context
rdf:value: We used SKBR3 as a model cell-line and compared its CGH profile with published [>>2<<,3,17] and our own manual hybridizations. Figure 1A represents the SKBR3 CGH profile published by Pollack et al., hybridized to a human cDNA micro array containing 6,691 different mapped human genes [2].
n2:mentions: n3:12297621

Subject Item: _:vb2203458
rdf:type: n2:Context
rdf:value: We used SKBR3 as a model cell-line and compared its CGH profile with published [2,>>3<<,17] and our own manual hybridizations. Figure 1A represents the SKBR3 CGH profile published by Pollack et al., hybridized to a human cDNA micro array containing 6,691 different mapped human genes [2].
n2:mentions: n3:16417655

Subject Item: _:vb2203459
rdf:type: n2:Context
rdf:value: Figure 1A represents the SKBR3 CGH profile published by Pollack et al., hybridized to a human cDNA micro array containing 6,691 different mapped human genes [>>2<<]. Figure 1B represents the SKBR3 CGH profile published by Shadeo and Lam., hybridized to a whole-genome tiling path BAC array containing 32,433 overlapping BAC-derived DNA segments [3]. Figure 1C represents the SKBR3 CGH profile published
n2:mentions: n3:12297621

Subject Item: _:vb2203460
rdf:type: n2:Context
rdf:value: Figure 1B represents the SKBR3 CGH profile published by Shadeo and Lam., hybridized to a whole-genome tiling path BAC array containing 32,433 overlapping BAC-derived DNA segments [>>3<<]. Figure 1C represents the SKBR3 CGH profile published by Jong et al., hybridized to a human oligonucleotide array containing 28,830 unique genes [17]. Figure 1D represents our manually hybridized SKBR3 CGH profile. Depicted in figure 1E
n2:mentions: n3:16417655

Subject Item: _:vb2203461
rdf:type: n2:Context
rdf:value: This figure summarizes all the breakpoints and estimated copy number levels as plotted in red in figure 1A–1F calculated by CGH-segmentation [>>16<<]. Breakpoint locations and calling of copy number levels (gain, unchanged, heterozygous loss, and homozygous loss) are provided as additional file 1 and 2. Although a lower density 3.5 k BAC array was used, figure 1G illustrates that
n2:mentions: n3:15705208

Subject Item: _:vb2203462
rdf:type: n2:Context
rdf:value: Although a lower density 3.5 k BAC array was used, figure 1G illustrates that nearly all aberrations and breakpoint in our results (figure 1D and 1E) are similar to the three published data sets [>>2<<,3,17]. We concluded that the dynamic range of both our manual and automated hybridization protocols are adequate to detect single copy number losses and gains. Reproducibility of this automated protocol is shown by replicate
n2:mentions: n3:12297621

Subject Item: _:vb2203463
rdf:type: n2:Context
rdf:value: Although a lower density 3.5 k BAC array was used, figure 1G illustrates that nearly all aberrations and breakpoint in our results (figure 1D and 1E) are similar to the three published data sets [2,>>3<<,17]. We concluded that the dynamic range of both our manual and automated hybridization protocols are adequate to detect single copy number losses and gains. Reproducibility of this automated protocol is shown by replicate hybridizations
n2:mentions: n3:16417655

Subject Item: _:vb2203464
rdf:type: n2:Context
rdf:value: To develop aCGH also as a diagnostic tool, it will be essential to validate its applicability on patient tumor samples and especially on archival FFPE tissue [>>18<<]. Extracted DNA from this material is often heavily cross-linked, heterogeneous (i.e. mix of cells of different genomic composition), fragmented, and rarely composed of 100% tumor cells.
n2:mentions: n3:16817944

Subject Item: _:vb2203465
rdf:type: n2:Context
rdf:value: For this particular tumor, the maximal CGH-segmentation [>>16<<] value was used as dynamic range ("Max CGHseg", table 5), because the homozygous loss on chromosome 11 would have a disproportional contribution to its value (same tumor as in figure 3).
n2:mentions: n3:15705208

Subject Item: _:vb2203466
rdf:type: n2:Context
rdf:value: Also the dynamic range kept at similar levels (again the highest ratio calculated by CGH-segmentation [>>16<<] was used because of the homologous loss in this tumor in chromosome 11 as depicted in figure 3).
n2:mentions: n3:15705208

Subject Item: _:vb2203467
rdf:type: n2:Context
rdf:value: Figure 4 shows the CGH profiles of the FFPE tumors (averaged log2 ratios of the manual and the automated hybridization), with very similar breakpoint locations and copy number estimates [>>16<<] for each hybridization method.
n2:mentions: n3:15705208

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