Whole Genome Amplification for Array Comparative Genomic Hybridization Using DNA Extracted from Formalin-Fixed, Paraffin-Embedded Histological Sections
2009; Elsevier BV; Volume: 11; Issue: 2 Linguagem: Inglês
10.2353/jmoldx.2009.080143
ISSN1943-7811
AutoresJian Huang, Jesse Pang, Takuya Watanabe, Ho‐Keung Ng, Hiroko Ohgaki,
Tópico(s)Cancer Genomics and Diagnostics
ResumoArray comparative genomic hybridization (CGH) is useful to assess genome-wide chromosomal imbalance, but the requirement for relatively large amounts of DNA can be a limitation, in particular for samples extracted from small tumor areas on paraffin sections. Whole genome amplification (WGA) can be performed before array CGH to obtain sufficient DNA, but the possibility of artifacts attributable to biased amplification cannot be excluded. We optimized the WGA protocol to generate sufficient DNA with minimum amplification bias. Using formalin-fixed, paraffin-embedded histological sections of tumors carrying known TP53 mutations, LOH 1p, LOH 10q, LOH 19q, and EGFR amplification, we first optimized the protocol so that these genetic alterations were detected after WGA. We found that a ligation step before WGA is important because it allows a short reaction time with Phi29 to generate WGA-DNA with greatly decreased amplification bias. Using template >150 ng of DNA, a ligation step before WGA, and a short reaction time with Phi29 DNA polymerase ( 4 μg) with minimum amplification bias (less than threefold). Using this protocol, we performed array CGH (Agilent 105K) before and after WGA. Pearson correlation analysis indicated a significant positive correlation in array CGH results between DNA before and after WGA (P < 0.0001). These results suggest that genetic analyses are possible using WGA-DNA extracted from paraffin sections, but that they should be performed with a carefully optimized and controlled protocol. Array comparative genomic hybridization (CGH) is useful to assess genome-wide chromosomal imbalance, but the requirement for relatively large amounts of DNA can be a limitation, in particular for samples extracted from small tumor areas on paraffin sections. Whole genome amplification (WGA) can be performed before array CGH to obtain sufficient DNA, but the possibility of artifacts attributable to biased amplification cannot be excluded. We optimized the WGA protocol to generate sufficient DNA with minimum amplification bias. Using formalin-fixed, paraffin-embedded histological sections of tumors carrying known TP53 mutations, LOH 1p, LOH 10q, LOH 19q, and EGFR amplification, we first optimized the protocol so that these genetic alterations were detected after WGA. We found that a ligation step before WGA is important because it allows a short reaction time with Phi29 to generate WGA-DNA with greatly decreased amplification bias. Using template >150 ng of DNA, a ligation step before WGA, and a short reaction time with Phi29 DNA polymerase ( 4 μg) with minimum amplification bias (less than threefold). Using this protocol, we performed array CGH (Agilent 105K) before and after WGA. Pearson correlation analysis indicated a significant positive correlation in array CGH results between DNA before and after WGA (P < 0.0001). These results suggest that genetic analyses are possible using WGA-DNA extracted from paraffin sections, but that they should be performed with a carefully optimized and controlled protocol. Array comparative genomic hybridization (CGH) is a useful method to detect amplifications and deletions at the whole-genome level and has contributed to the detection of novel gene loci associated with pathogenesis of various human neoplasms.1Pinkel D Segraves R Sudar D Clark S Poole I Kowbel D Collins C Kuo WL Chen C Zhai Y Dairkee SH Ljung BM Gray JW Albertson DG High resolution analysis of DNA copy number variation using comparative genomic hybridization to microarrays.Nat Genet. 1998; 20: 207-211Crossref PubMed Scopus (1774) Google Scholar2Veltman JA Fridlyand J Pejavar S Olshen AB Korkola JE DeVries S Carroll P Kuo WL Pinkel D Albertson D Cordon-Cardo C Jain AN Waldman FM Array-based comparative genomic hybridization for genome-wide screening of DNA copy number in bladder tumors.Cancer Res. 2003; 63: 2872-2880PubMed Google Scholar3Paris PL Albertson DG Alers JC Andaya A Carroll P Fridlyand J Jain AN Kamkar S Kowbel D Krijtenburg PJ Pinkel D Schroder FH Vissers KJ Watson VJ Wildhagen MF Collins C van Dekken H High-resolution analysis of paraffin-embedded and formalin-fixed prostate tumors using comparative genomic hybridization to genomic microarrays.Am J Pathol. 2003; 162: 763-770Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar However, array CGH requires a relatively large amount of DNA (>2 μg), and this has been a major limitation impeding application of array CGH to certain samples, in particular DNA extracted from small tumor areas or from single-cell, laser-captured samples on formalin-fixed, paraffin-embedded sections. To overcome this limitation, whole genome amplification (WGA) methods have been developed. Previously, most WGA methods were polymerase chain reaction (PCR)-based (using Taq polymerase), such as degenerated oligonucleotide-primed,4Daigo Y Chin SF Gorringe KL Bobrow LG Ponder BA Pharoah PD Caldas C Degenerate oligonucleotide primed-polymerase chain reaction-based array comparative genomic hybridization for extensive amplicon profiling of breast cancers: a new approach for the molecular analysis of paraffin-embedded cancer tissue.Am J Pathol. 2001; 158: 1623-1631Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar,5Cheung VG Nelson SF Whole genome amplification using a degenerate oligonucleotide primer allows hundreds of genotypes to be performed on less than one nanogram of genomic DNA.Proc Natl Acad Sci USA. 1996; 93: 14676-14679Crossref PubMed Scopus (223) Google Scholar ligation-mediated,6Guillaud-Bataille M Valent A Soularue P Perot C Inda MM Receveur A Smaili S Roest CH Benard J Bernheim A Gidrol X Danglot G Detecting single DNA copy number variations in complex genomes using one nanogram of starting DNA and BAC-array CGH.Nucleic Acids Res. 2004; 32: e112Crossref PubMed Scopus (36) Google Scholar and primer extension preamplification7Mill J Yazdanpanah S Guckel E Ziegler S Kaminsky Z Petronis A Whole genome amplification of sodium bisulfite-treated DNA allows the accurate estimate of methylated cytosine density in limited DNA resources.Biotechniques. 2006; 41: 603-607Crossref PubMed Scopus (27) Google Scholar PCR procedures. However, several studies have suggested that these methods may generate significant sequence representation bias and artifacts during the WGA process.8Pinard R de Winter A Sarkis GJ Gerstein MB Tartaro KR Plant RN Egholm M Rothberg JM Leamon JH Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing.BMC Genomics. 2006; 7: 216Crossref PubMed Scopus (243) Google Scholar More recently, a non-PCR-based isothermal method, multiple displacement amplification, has been applied to small DNA samples, leading to the synthesis of DNA with less sequence representation bias.9Lizardi PM Huang X Zhu Z Bray-Ward P Thomas DC Ward DC Mutation detection and single-molecule counting using isothermal rolling-circle amplification.Nat Genet. 1998; 19: 225-232Crossref PubMed Scopus (1099) Google Scholar,10Dean FB Hosono S Fang L Wu X Faruqi AF Bray-Ward P Sun Z Zong Q Du Y Du J Driscoll M Song W Kingsmore SF Egholm M Lasken RS Comprehensive human genome amplification using multiple displacement amplification.Proc Natl Acad Sci USA. 2002; 99: 5261-5266Crossref PubMed Scopus (1091) Google Scholar This method is based on the annealing of random hexamers to denatured DNA, followed by strand-displacement synthesis at a constant temperature, resulting in DNA products of high molecular weight.11Blanco L Bernad A Lazaro JM Martin G Garmendia C Salas M Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication.J Biol Chem. 1989; 264: 8935-8940Abstract Full Text PDF PubMed Google Scholar The reaction is catalyzed by Phi29 DNA polymerase or a large fragment of the Bst DNA polymerase, which results in error rates 100 times lower than with Taq polymerase.12Eckert KA Kunkel TA DNA polymerase fidelity and the polymerase chain reaction.PCR Methods Appl. 1991; 1: 17-24Crossref PubMed Scopus (352) Google Scholar,13Esteban JA Salas M Blanco L Fidelity of phi 29 DNA polymerase. Comparison between protein-primed initiation and DNA polymerization.J Biol Chem. 1993; 268: 2719-2726Abstract Full Text PDF PubMed Google Scholar Spits and colleagues14Spits C Le Caignec C De Rycke M Van Haute L Van Steirteghem A Liebaers I Sermon K Optimization and evaluation of single-cell whole-genome multiple displacement amplification.Hum Mutat. 2006; 27: 496-503Crossref PubMed Scopus (122) Google Scholar compared Phi29 and Bst DNA polymerases, and showed that the Phi29 generated accurately sufficient DNA, whereas the Bst showed low efficiency and high error rate during WGA. Most of the WGA studies recently published use Phi29.8Pinard R de Winter A Sarkis GJ Gerstein MB Tartaro KR Plant RN Egholm M Rothberg JM Leamon JH Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing.BMC Genomics. 2006; 7: 216Crossref PubMed Scopus (243) Google Scholar,10Dean FB Hosono S Fang L Wu X Faruqi AF Bray-Ward P Sun Z Zong Q Du Y Du J Driscoll M Song W Kingsmore SF Egholm M Lasken RS Comprehensive human genome amplification using multiple displacement amplification.Proc Natl Acad Sci USA. 2002; 99: 5261-5266Crossref PubMed Scopus (1091) Google Scholar,14Spits C Le Caignec C De Rycke M Van Haute L Van Steirteghem A Liebaers I Sermon K Optimization and evaluation of single-cell whole-genome multiple displacement amplification.Hum Mutat. 2006; 27: 496-503Crossref PubMed Scopus (122) Google Scholar15Uda A Tanabayashi K Fujita O Hotta A Yamamoto Y Yamada A Comparison of whole genome amplification methods for detecting pathogenic bacterial genomic DNA using microarray.Jpn J Infect Dis. 2007; 60: 355-361PubMed Google Scholar16Hosono S Faruqi AF Dean FB Du Y Sun Z Wu X Du J Kingsmore SF Egholm M Lasken RS Unbiased whole-genome amplification directly from clinical samples.Genome Res. 2003; 13: 954-964Crossref PubMed Scopus (328) Google Scholar17Lage JM Leamon JH Pejovic T Hamann S Lacey M Dillon D Segraves R Vossbrinck B Gonzalez A Pinkel D Albertson DG Costa J Lizardi PM Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH.Genome Res. 2003; 13: 294-307Crossref PubMed Scopus (213) Google Scholar Several studies have demonstrated that WGA using Phi29 DNA polymerase yielded significantly less amplification bias than PCR-based methods,8Pinard R de Winter A Sarkis GJ Gerstein MB Tartaro KR Plant RN Egholm M Rothberg JM Leamon JH Assessment of whole genome amplification-induced bias through high-throughput, massively parallel whole genome sequencing.BMC Genomics. 2006; 7: 216Crossref PubMed Scopus (243) Google Scholar,10Dean FB Hosono S Fang L Wu X Faruqi AF Bray-Ward P Sun Z Zong Q Du Y Du J Driscoll M Song W Kingsmore SF Egholm M Lasken RS Comprehensive human genome amplification using multiple displacement amplification.Proc Natl Acad Sci USA. 2002; 99: 5261-5266Crossref PubMed Scopus (1091) Google Scholar,15Uda A Tanabayashi K Fujita O Hotta A Yamamoto Y Yamada A Comparison of whole genome amplification methods for detecting pathogenic bacterial genomic DNA using microarray.Jpn J Infect Dis. 2007; 60: 355-361PubMed Google Scholar and validated the use of Phi29 DNA polymerase for WGA on high-quality genomic DNA extracted from 1 to 1000 cells.14Spits C Le Caignec C De Rycke M Van Haute L Van Steirteghem A Liebaers I Sermon K Optimization and evaluation of single-cell whole-genome multiple displacement amplification.Hum Mutat. 2006; 27: 496-503Crossref PubMed Scopus (122) Google Scholar,16Hosono S Faruqi AF Dean FB Du Y Sun Z Wu X Du J Kingsmore SF Egholm M Lasken RS Unbiased whole-genome amplification directly from clinical samples.Genome Res. 2003; 13: 954-964Crossref PubMed Scopus (328) Google Scholar,17Lage JM Leamon JH Pejovic T Hamann S Lacey M Dillon D Segraves R Vossbrinck B Gonzalez A Pinkel D Albertson DG Costa J Lizardi PM Whole genome analysis of genetic alterations in small DNA samples using hyperbranched strand displacement amplification and array-CGH.Genome Res. 2003; 13: 294-307Crossref PubMed Scopus (213) Google Scholar In contrast, only a few studies have successfully applied multiple displacement amplification-based WGA with DNA from formalin-fixed, paraffin-embedded tissues,18Aviel-Ronen S Qi Zhu C Coe BP Liu N Watson SK Lam WL Tsao MS Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues.BMC Genomics. 2006; 7: 312Crossref PubMed Scopus (36) Google Scholar19Devries S Nyante S Korkola J Segraves R Nakao K Moore D Bae H Wilhelm M Hwang S Waldman F Array-based comparative genomic hybridization from formalin-fixed, paraffin-embedded breast tumors.J Mol Diagn. 2005; 7: 65-71Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar20Bredel M Bredel C Juric D Kim Y Vogel H Harsh GR Recht LD Pollack JR Sikic BI Amplification of whole tumor genomes and gene-by-gene mapping of genomic aberrations from limited sources of fresh-frozen and paraffin-embedded DNA.J Mol Diagn. 2005; 7: 171-182Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar in which the DNA is degraded because of formalin fixation, containing strand breaks, base damage, and DNA-protein crosslinks.21Lehmann U Kreipe H Real-time PCR analysis of DNA and RNA extracted from formalin-fixed and paraffin-embedded biopsies.Methods. 2001; 25: 409-418Crossref PubMed Scopus (316) Google Scholar,22Lewis F Maughan NJ Smith V Hillan K Quirke P Unlocking the archive—gene expression in paraffin-embedded tissue.J Pathol. 2001; 195: 66-71Crossref PubMed Scopus (261) Google Scholar BAC array CGH showed similar results before and after eightfold WGA with Klenow enzyme, when 50 ng of template DNA extracted from paraffin sections was used,19Devries S Nyante S Korkola J Segraves R Nakao K Moore D Bae H Wilhelm M Hwang S Waldman F Array-based comparative genomic hybridization from formalin-fixed, paraffin-embedded breast tumors.J Mol Diagn. 2005; 7: 65-71Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar but the amounts of amplified DNA were too small to be applied to recent commercially available platforms. In one study with 10 ng of template DNA extracted from paraffin sections, WGA using Bst DNA polymerase produced a median 990-fold DNA amplification, but 27 K BAC array CGH showed that most of the deletions observed in non-WGA DNA were not reproduced after WGA, although representational distortion of threefold was achieved by quantitative PCR analysis in six genes.18Aviel-Ronen S Qi Zhu C Coe BP Liu N Watson SK Lam WL Tsao MS Large fragment Bst DNA polymerase for whole genome amplification of DNA from formalin-fixed paraffin-embedded tissues.BMC Genomics. 2006; 7: 312Crossref PubMed Scopus (36) Google Scholar Bredel and colleagues20Bredel M Bredel C Juric D Kim Y Vogel H Harsh GR Recht LD Pollack JR Sikic BI Amplification of whole tumor genomes and gene-by-gene mapping of genomic aberrations from limited sources of fresh-frozen and paraffin-embedded DNA.J Mol Diagn. 2005; 7: 171-182Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar reported that WGA-DNA from paraffin sections could be used for array CGH using Phi29, but normalization using amplified reference DNA was necessary because of significant amplification bias. The objective of the present study was to establish a Phi29-based WGA method using small amounts of DNA extracted from formalin-fixed, paraffin-embedded tissues for array CGH. We have optimized several critical steps including DNA extraction, amount of template DNA, and reaction time with Phi29. Because one of the key factors leading to failure of unbiased WGA on DNA extracted from paraffin sections may be the presence of highly degraded DNA with short fragments, we also assessed whether a ligation step before WGA improves the results. To validate WGA, we used tumor samples on paraffin sections, in which known TP53 mutations, LOH 1p, LOH 10q, LOH 19q, and EGFR amplification were present. Genetic alterations in two tumor samples (glioblastomas) on formalin-fixed, paraffin-embedded histological sections were previously reported.23Ohgaki H Dessen P Jourde B Horstmann S Nishikawa T Di Patre PL Burkhard C Schuler D Probst-Hensch NM Maiorka PC Baeza N Pisani P Yonekawa Y Yasargil MG Lutolf UM Kleihues P Genetic pathways to glioblastoma: a population-based study.Cancer Res. 2004; 64: 6892-6899Crossref PubMed Scopus (1042) Google Scholar One sample (tumor 1) had LOH 1p, EGFR amplification, and a TP53 mutation (GTT→CTT, Val274Leu in exon 8) and another (tumor 2) had LOH 1p, LOH 10q, LOH 19q, and a TP53 mutation (ATG→ATA, Met237Ile in exon 7). Both tumor samples were fixed in buffered formalin and embedded in paraffin more than 15 years ago. Paraffin sections were cut to 4 to 5 μm thickness 5 years ago, and stored at room temperature. Using these samples, we assessed several critical steps mentioned below, to ensure that these genetic alterations were reproducibly detected after WGA. Paraffin sections were deparaffinized in xylene for 15 minutes and then put into 100%, 95%, and 70% ethanol for 5 minutes each and then into distilled water. After air-drying, tumor areas on the sections were scraped off the slide and washed with PBS (pH 7.4) solution twice, suspended in 400 μl of 1 N NaSCN solution, and incubated at 37°C overnight. Samples were then suspended in 400 μl of DNA extraction buffer (mixture of 360 μl of ATL buffer and 40 μl of proteinase K; DNeasy mini kit, Qiagen, Valencia, CA), and were incubated at 55°C overnight. Additional proteinase K (80 μl) was added and incubated for a total of 60 hours. After reaction with 8 μl of RNase (100 mg/ml) for 2 minutes at room temperature, the samples were made up with 420 μl of ATL to a total volume of 900 μl and separated into two parts (450 μl each). Each part was mixed with 450 μl of AL buffer and 450 μl of 100% ethanol, and incubated at room temperature for 5 minutes. The samples were then loaded onto DNeasy mini spin columns (DNeasy mini kit, Qiagen, CA). The column was washed with AW1 buffer and the column membrane dried with 80% ethanol, after which the purified genomic DNA was eluted with 25 μl of nuclease-free water. The DNA concentration was determined by spectrophotometric absorption at 230, 260, and 280nm and the quality was calculated as the A260/A230 and A260/A280 ratio. Genomic DNA (10, 50, 150, 300 ng) samples were ligated at 24°C for 30 minutes with ligase, and then amplified with Phi29 enzyme at 30°C for 30, 60, 90, or 180 minutes. Other samples were not ligated before WGA. Ligation and amplification with Phi29 enzyme were performed using components of a Qiagen FFPE amplification kit. Briefly, various amounts of purified genomic DNA in a total volume of 10 μl were heated to 95°C for 5 minutes for denaturation. After cooling on ice for 5 minutes, 8 μl of FFPE buffer, 1 μl of ligation enzyme, and 1 μl of FFPE enzyme were added, and then incubated at 24°C for 30 minutes, followed by heat inactivation at 95°C for 5 minutes. After ligation, 20-μl samples were mixed with 30 μl of a prepared reaction mixture (29 μl of reaction buffer and 1 μl of Midi Phi29 enzyme per reaction), and were incubated at 30°C for different reaction times (30 to 180 minutes). After amplification, the Phi29 enzyme was inactivated by heating at 95°C for 10 minutes. To identify TP53 mutations in exon 7 or exon 8 in WGA products, PCR amplification was performed as previously described.24Huang J Grotzer MA Watanabe T Hewer E Pietsch T Rutkowski S Ohgaki H Mutations in the Nijmegen breakage syndrome gene in medulloblastomas.Clin Cancer Res. 2008; 14: 4053-4058Crossref PubMed Scopus (24) Google Scholar Briefly, PCR was performed in a total volume of 10 μl, consisting of 1 μl each of WGA products (concentration, ∼100 ng/μl), 0.5 U of Platinum TaqDNA polymerase (Invitrogen, Cergy Pontoise, France), 1 mmol/L MgCl2, 0.1 mmol/L of each dNTP, 0.2 mmol/L of each primer, 10 mmol/L Tris-HCl, pH 8.3, and 50 mmol/L KCl in a thermal cycler (Biometra, Archamps, France) with an initial denaturing step at 95°C for 5 minutes, followed by 37 cycles of denaturation at 95°C for 50 seconds, annealing at 60°C (exon 7) or 61°C (exon 8) for 60 seconds, extension at 72°C for 60 seconds, and a final extension at 72°C for 5 minutes. The sequencing reaction was performed using a Big Dye Terminator cycle sequencing kit (ABI Prism; Applied Biosystems, Foster City, CA) in an ABI 3100 Prism DNA sequencer (Applied Biosystems). LOH analysis was performed using two microsatellite markers (D10S536, D10S1683) on chromosome 10q, two (D19S408, D10S596) on chromosome 19q, and two (D1S2736, D1S468) on chromosome 1p. For markers D1S2736 and D19S408, PCR reactions were performed in a total volume of 12.5 μl with 6.25 μl of 2× TaqMan Universal PCR Master Mix (Applied Biosystems), 4 μl of primer sets (1.25 μmol/L of each primer), 1.25 μl of 1.5 μmol/L probe [21-bp oligomer complementary to the microsatellite CA repeat: 5′,6-carboxyfluorescein (FAM)-TGTGTGTGTGTGTGTGTGTGT-3′,6-carboxy-tetramethylrhodamine], and 10 ng of DNA. For markers D10S536, D10S1683, D1S468, D19S596, PCR reactions were performed in a total volume of 18.75 μl with 9.375 μl of 2× AmpliTaq Gold PCR Master Mix (Roche, Basel, Switzerland), 6 μl of primer sets (2.5 μmol/L of each primer), 1.875 μl of 1.5 μmol/L of the same probe as above, and 20 ng of DNA, with cycling parameters as reported previously.25Nigro JM Takahashi MA Ginzinger DG Law M Passe S Jenkins RB Aldape K Detection of 1p and 19q loss in oligodendroglioma by quantitative microsatellite analysis, a real-time quantitative polymerase chain reaction assay.Am J Pathol. 2001; 158: 1253-1262Abstract Full Text Full Text PDF PubMed Scopus (155) Google Scholar PCR was performed for each individual DNA sample in triplicate on a 96-well optical plate with an ABI 7900HT PCR system (Applied Biosystems). Amplification of a pool of six reference loci served to normalize for differences in the amount of total input DNA, as described previously. To calculate the average δCt [δCT (normal)], DNA was isolated from 10 formalin-fixed, paraffin-embedded normal tissues. The Ct, δCt [Ct (microsatellite) − Ct (reference pool)], δδCt [δCt (tumor) − δCt (normal)] values, the relative copy number (2−δδCt), and the tolerance interval with confidence of 95% determined from the pooled SD of normal DNA for the loci were calculated as reported previously. On the basis of this tolerance interval, copy numbers 500 bp) were excised, and NT1 buffer was added (300 μl/100 mg agarose gel). Then NucleoTraPCR suspension was added (4 μl/μg of DNA) and the sample was incubated at 50°C until the gel slices were dissolved. The sample was then centrifuged for 30 seconds at 10,000 × g and the supernatant was discarded. Pellets were washed with 400 μl of buffers NT2, NT3, and NT3, respectively. The silica matrix was dried at room temperature for 15 minutes. Nuclease-free water (30 μl) was added to the pellet, which was resuspended by stirring. The mixture was incubated at 55°C for 15 minutes. Samples were then centrifuged at 10,000 × g for 30 seconds and supernatants were transferred to clean tubes. The yield of WGA products before and after purification was determined by the PicoGreen dsDNA method (Invitrogen, CA) and UV absorption method. The genomic profile changes of paired DNA samples before and after WGA were compared using a 105K CGH oligonucleotide microarrray (Agilent Technologies, Santa Clara, CA) according to the manufacturer's instructions. Briefly, 1 to 2 μg of sample and sex-matched reference DNA were chemically labeled, respectively, with ULS-Cy5 and ULS-Cy3 at 85°C for 30 minutes (Oligo aCGH labeling kit for FFPE samples, Agilent). Labeled samples were purified (Genomic DNA purification module, Agilent), combined, mixed with human Cot-1 DNA, and denatured at 95°C (Oligo aCGH hybridization kit). The mixture was applied to microarrays and hybridization was performed at 65°C for 40 hours. After hybridization, the microarrays were washed in Oligo aCGH wash buffer 1 at room temperature for 5 minutes and wash buffer 2 at 37°C for 1 minute. After drying, the microarrays were scanned using a DNA microarray scanner G2565BA (Agilent) and data (log2) were extracted from raw microarray image files using Feature Extraction software (version 9; Agilent). Data were analyzed by DNA Analytics software (version 4.0; Agilent) with default filter settings. The aberration detection method 2 (ADM2) algorithm with centralization and fuzzy zero correction was used to define aberrant intervals. Each of the arrays was independently analyzed and evaluated for genetic alterations before and after WGA amplification. To assess the similarity between DNA with or without WGA, Pearson's correlations of log2 ratios of probes from autosomes among tumor 1 and tumor 2 samples were performed with Graphpad Prism software (GraphPad, La Jolla, CA). WGA produced DNA of high molecular weight (0.5 ∼ 20 kb; Figure 1). The negative control (H2O) did not generate any DNA product when samples were incubated with Phi29 for <1.5 hours (Figure 1). A ligation step before WGA significantly affected the results. Samples treated with a ligation s
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