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Application Note Use of BAC aCGH for molecular karyotyping of whole genome amplified FFPE DNA samples Christopher Williams, Product Specialist, PerkinElmer Life and Analytical Sciences Steve Michalik, Product Manager, Sigma-Aldrich Biotechnology Introduction Archival, formalin-fixed, paraffin-embedded (FFPE) tissues represent an invaluable source of material for molecular genetic studies. There is a growing desire to profile FFPE samples via array comparative genomic hybridization (aCGH), however many of these samples are unsuitable for microarray analysis due to nucleic acid damage introduced during the fixation process. Several methods have been shown to be effective in the assessment of DNA quality from sources of damaged DNA, with PCR-based assays being the most widely accepted (1, 2, 3). We apply a simple, gel-based, multiplex PCR assay to FFPE tissues prior to whole genome amplification (WGA) and aCGH analysis to assess DNA quality and predict aCGH feasibility. In addition, there are currently two commercially available technologies for the application of WGA. One is Sigma’s GenomePlex WGA product line that is based on a modified DOP-PCR technology (4), and the other is multiple displacement amplification (MDA) WGA technology (5). Several groups have applied MDA-based WGA to FFPE samples, but the requirement of full-length template DNA appears to preclude this method from use with archived samples (6, 7, 8). Here we show that Sigma’s GenomePlex WGA platform and PerkinElmer Spectral Chip™ 2600 BAC array platform work effectively for generating high quality aCGH data from archival samples when starting with 10ng FFPE genomic DNA or ~1mg FFPE tissue. FFPE tissue can be used directly with the GenomePlex Tissue Whole Genome Amplification Kit and analyzed for chromosomal gains or losses via PerkinElmer Spectral Chip™ 2600 BAC arrays to generate high quality aCGH data. The Spectral Chip™ 2600 1 Mb BAC Array Platform Array-based comparative genomic hybridization (aCGH) yields data that is analogous to traditional karyotyping but with significantly greater throughput, resolution and ease of use. Traditional karyotyping methods for detecting genomic variation, such as FISH (Fluorescence in situ Hybridization) or G-banding are well established for detecting chromosomal aberrations, but they do not provide the speed, throughput, or resolution required for today’s cytogenetic and research laboratories. The SpectralChip™ 2600 array allows the researcher to perform the equivalent, of thousands of FISH experiments simultaneously in a 24-hour protocol. Additionally, the SpectralChip™ array provides greater resolution and improved data analysis tools. www.perkinelmer.com The 2605 non-overlapping bacterial artificial chromosome (BAC) clones on the SpectralChip™ array span the entire genome. The BAC clones are spaced at approximately 1 megabase intervals, as compared to traditional karyotyping, that has approximately 10 megabase resolution. The clones are covalently coupled to glass microscope slides, spotted in duplicate. Sample and reference DNA are labeled in a two color, ratiometric experiment. A fluorescent scanner captures data from the array and SpectralWare® software converts the scanner output data into an intensity ratio profile. The software analyzes copy number changes and displays the location of the changes within the genome. The aCGH process combines the whole genome perspective of chromosome karyotypmg with the increased resolution of FISH and the high throughput of DNA arrays. The SpectralChip™ array can generate molecular profiles in a single experiment. Hybridization can be completed in 16 hours, and data are available in </=24 hours. Methods FFPE tissues were WGA amplified directly using Sigma’s GenomePlex Tissue WGA kit (Cat # WGA5), whereas purified genomic DNA was WGA amplified using Sigma’s GenomePlex WGA kit (Cat # WGA2) per manufacturer’s recommendations. BAC aCGH profiles were generated using 1µg WGA DNA on PerkinElmer Spectral Chip™ 2600 BAC array (Cat # 4027-0020) platform. Array CGH data was analyzed using PerkinElmer SpectralWare® aCGH Analysis Software (Cat # 5007-1010) using default settings. Oligo aCGH was performed by a certified service provider (UHN Microarray center, Toronto, Canada). Gel-based multiplex PCR assay for DNA quality assessment A single reaction, multiplex PCR assay containing a mixture of five primer sets was used to determine FFPE DNA quality and WGA/aCGH outcome. High quality FFPE DNA produces all five amplicons, whereas lower quality DNA produces no amplicons, or a subset of the amplicons (figures 1 to 3). Figure 1. Primer information for multiplex PCR assay. Primer sequences were derived from the UniSTS database, and the UniSTS accession numbers, primer sequences, amplicon sizes, and chromosomal locations of the amplicons are provided. 2 Figure 2. Gel of multiplex amplicons generated from FFPE lysates. Approximately 1mg of tissue was collected from five FFPE tissue samples followed by processing with the GenomePlex Tissue Whole Genome Amplification Kit (Sigma Cat. WGA5) as outlined in the technical bulletin. 5 µl of undiluted WGA5 tissue lysate was subjected to multiplex PCR amplification as described at http://www.sigmaaldrich.com/ img/assets/3090/Advisor_FFPE_DNA_ diagnostic.pdf, and 5 µl of each reaction was resolved on a 4% agarose gel. All five bands were amplified in lanes 1 and 2 indicating that these FFPE tissue lysates contain high quality genomic DNA, whereas lanes 3, 4 and 5 contain low quality DNA since all, or most, of the multiplex PCR fragments were not amplifiable. Similar results were observed when purified DNA or amplified WGA product derived from these FFPE tissues were used directly in the multiplex qPCR assay (data not shown). Figure 3. aCGH of results derived from Samples 1 and 5. Amplified DNA (1ug) from samples 1 and 5 were analyzed using the PerkinElmer BAC aCGH platform. Sample 1 shows a normal X chromosome profile as predicted by the multiplex PCR assay (Figure 2), whereas Sample 5 shows a very poor X chromosome profile due to low quality DNA predicted from the multiplex PCR assay. 3 Validation of GenomePlex Tissue WGA Kit and Spectral Chip 2600 BAC aCGH platform using a model system We generated a series of GenomePlex amplified aCGH profiles from male and female FFPE tissues and female DNA known to contain gains on the X-chromosome, and hybridized these against GenomePlex amplified normal female control DNA from Promega. Briefly, FFPE tissue or genomic DNA was whole genome amplified per the Sigma product bulletins (Sigma Cat# WGA5 and WGA2, respectively). 1µg of resulting products was used for BAC aCGH analysis using PerkinElmer Labeling Reagents (Cat # 4040-0020), Hybridization Reagents (Cat # 4050-0010), Spectral Chip™ 2600 BAC array and Wash Buffer Pack (Cat # 40380020) per manufacturer’s recommendations. (Figures 4 to 7) The ideograms generated using PerkinElmer SpectralWare® aCGH analysis software below show varying X and Y chromosome dosage effects in amplified aCGH. The genotypes for each sample and reference are given in the figure legends, whereas array metrics are provided in the tables below each plot. Mixed male and female DNA samples (1:1) are indicated as XY+XX, whereas chromosome trisomy X DNA is indicated as XXX. Figure 4. GenomePlex Amplified XY vs XX (1:2 loss of X; 1:0 gain of Y). 4 Figure 5. GenomePlex Amplified XY vs XY (1:1 ratio of X and Y). Figure 6. GenomePlex Amplified XXX vs XX (3:2 gain of X). 5 Figure 7. GenomePlex Amplified XY +XX vs XX (1.5:2 dilution of X). FFPE DNA analysis via BAC aCGH and oligo aCGH aCGh profiles were generated using the PerkinElmer Spectral Chip 266 BAC array platform and using an established oligo based array product. The BAC arrays were performed using the supplied manufacturers protocol and the oligo arrays were performed by a certified service provider. Figures 8-11 illustrate the design and results of experiments performed to compare results using fresh frozen and FFPE multiple myeloma samples. Figure 8. Diagram of the experimental workflow comparing PerkinElmer BAC aCGH and oligo aCGH analysis of GenomePlex amplified paired fresh frozen and FFPE myloma samples and GenomePlex amplified control DNA (Promega Corp.). Hybridizations were performed against control DNA of the opposite sex. All hybridizations were performed in quadruplicate. 6 Figure 9. SpectralChip 2600 ideograms of chromosome 7 comparing paired frozen and FFPE myeloma samples. The whole genome data shown here was generated using PerkinElmer SpectralWare® aCGH Analysis Software. These whole genome results of chromosome 7 are from 1 representative sample, and they show excellent concordance between fresh frozen and FFPE samples. Figure 10. Oligo aCGH of frozen and FFPE multiple myeloma samples. Representative ideograms of comparison data from four sample hybridizations on chromosomes 12, 15, 18, and 22 are shown. When comparing the ”Fresh” and ”FFPE” samples there is little or no correlation between the samples. 7 Figure 11. Oligo array and BAC array ideograms of chromosome 7 comparing results from fresh frozen and FFPE multiple myeloma samples. Note the high correlation of data in the BAC array results and the low level of correlation in the oligo array data. Conclusion • • • • FFPE samples can now be a valuable source of archival material for genomic data analysis DNA is extracted and WGA amplified directly from tissue with Sigma’s optimized WGA5 kit DNA quality is predicted with a simple, gel-based, multiplex PCR test and linked to aCGH success SpectralChip™ 2600 BAC array data correlates well when comparing fresh frozen samples and FFPE samples • Oligo-probe aCGH data did not correlate well when comparing fresh frozen samples and FFPE samples prepared in the same manner. References (general) 1) E.H. Van Beers et al. A multiplex PCR predictor for aCGH success of FFPE samples. Br. J. Cancer, 2006, 94:333-337. 2) H.M. Hansen et al. DNA quantification of whole genome amplified samples for genotyping on a multiplexed bead array platform. Cancer Epidemiol. Biomarkers Prev. 2007, 16:1686–1690. 3) F. Wang et al. DNA degradation test predicts success in whole genome amplification from diverse clinical samples. J. Mol. Diag. 2007, 9:441-451. 4) E. Mueller. Genomic analysis of formalin-fixed paraffin embedded (FFPE) tissues through the use of whole genome amplification (WGA). http://www.sigmaaldrich.com/sigma/general%20information /ffpewhitepaper. October, 2007. 5) F.B. Dean et al. Rapid Amplification of Plasmid and Phage DNA Using Phi29 DNA Polymerase and Multiply-Primed Rolling Circle Amplification. Genome Res, 2001, 11:1095-1099. 6) S.E. Little et al. Array CGH using whole genome amplification of fresh-frozen and formalin-fixed paraffin-embedded tumor DNA. Genomics, 2006, 87:298-306. 7) K. Iwamoto et al. Evaluation of whole genome amplification methods using postmortem brain samples. J. Neuroscience Methods, 2007, 165:104-110. 8) M.I.L. Sjoholm et al. Comparison of archival plasma and formalin-fixed paraffin-embedded tissue for genotyping in hepatocellular carcinoma. Cancer Epidemiol. Biomarkers Prev, 2005, 14:251-255. PerkinElmer, Inc. 940 Winter Street Waltham, MA 02451 USA Phone: (800) 762-4000 or (+1) 203-925-4602 www.perkinelmer.com PerkinElmer Genetic Screening Center of Excellence Wallac Oy, PO Box 10 20101 Turku, Finland Phone: + 358 2 2678 111 Fax: + 358 2 2678 357 For a complete listing of our global offices, visit www.perkinelmer.com/lasoffices ©2008 PerkinElmer, Inc. All rights reserved. Spectral Chip is a trademark and Scan Array, SpectralWare and PerkinElmer are registered trademarks of PerkinElmer, Inc. All trademarks depicted are the property of their respective holders or owners. PerkinElmer reserves the right to change this document at any time and disclaims liability for editorial, pictorial or typographical errors. All PerkinElmer diagnostic products may not be available in all countries. For information on availability please contact your local representative. 1244-9857, June 2008 Printed in Finland by Offset House Oy Naantali 2008 www.perkinelmer.com