DNA Degradation Test Predicts Success in Whole-Genome Amplification from Diverse Clinical Samples
2007; Elsevier BV; Volume: 9; Issue: 4 Linguagem: Inglês
10.2353/jmoldx.2007.070004
ISSN1943-7811
AutoresFengfei Wang, Xin Lin, Christine Briggs, Ewa Sicińska, Sandra M. Gaston, Harvey J. Mamon, Matthew H. Kulke, Raffaella Zamponi, Massimo Loda, Elizabeth A. Maher, Shuji Ogino, Charles S. Fuchs, Jin Li, Carlos Hader, G. Mike Makrigiorgos,
Tópico(s)Cancer Genomics and Diagnostics
ResumoThe need to apply modern technologies to analyze DNA from diverse clinical samples often stumbles on suboptimal sample quality. We developed a simple approach to assess DNA fragmentation in minute clinical samples of widely different origin and the likelihood of success of degradation-tolerant whole genome amplification (restriction and circularization-aided rolling circle amplification, RCA-RCA) and subsequent polymerase chain reaction (PCR). A multiplex PCR amplification of four glyceraldehyde-3-phosphate dehydrogenase amplicons of varying sizes was performed using genomic DNA from clinical samples, followed by size discrimination on agarose gel or fluorescent denaturing high-performance liquid chromatography (dHPLC). RCA-RCA followed by real-time PCR was also performed, for correlation. Even minimal quantities of longer PCR fragments (∼300 to 400 bp), visible via high-sensitivity fluorescent dHPLC or agarose gel, were essential for the success of RCA-RCA and subsequent PCR-based assays. dHPLC gave a more accurate correlation between DNA fragmentation and sample quality than agarose gel electrophoresis. Multiplex-PCR-dHPLC predicted correctly the likelihood of assay success in formalin-fixed, paraffin-embedded samples fixed under controlled conditions and of different ages, in laser capture microdissection samples, in tissue print micropeels, and plasma-circulating DNA. Estimates of the percent information retained relative to snap-frozen DNA are derived for real-time PCR analysis. The assay is rapid and convenient and can be used widely to characterize DNA from any clinical sample of unknown quality. The need to apply modern technologies to analyze DNA from diverse clinical samples often stumbles on suboptimal sample quality. We developed a simple approach to assess DNA fragmentation in minute clinical samples of widely different origin and the likelihood of success of degradation-tolerant whole genome amplification (restriction and circularization-aided rolling circle amplification, RCA-RCA) and subsequent polymerase chain reaction (PCR). A multiplex PCR amplification of four glyceraldehyde-3-phosphate dehydrogenase amplicons of varying sizes was performed using genomic DNA from clinical samples, followed by size discrimination on agarose gel or fluorescent denaturing high-performance liquid chromatography (dHPLC). RCA-RCA followed by real-time PCR was also performed, for correlation. Even minimal quantities of longer PCR fragments (∼300 to 400 bp), visible via high-sensitivity fluorescent dHPLC or agarose gel, were essential for the success of RCA-RCA and subsequent PCR-based assays. dHPLC gave a more accurate correlation between DNA fragmentation and sample quality than agarose gel electrophoresis. Multiplex-PCR-dHPLC predicted correctly the likelihood of assay success in formalin-fixed, paraffin-embedded samples fixed under controlled conditions and of different ages, in laser capture microdissection samples, in tissue print micropeels, and plasma-circulating DNA. Estimates of the percent information retained relative to snap-frozen DNA are derived for real-time PCR analysis. The assay is rapid and convenient and can be used widely to characterize DNA from any clinical sample of unknown quality. Archival specimens represent a vast resource for discovery and evaluation of prognostic DNA markers. In the United States alone, there are more than 300 million archived tissue samples with ∼20 million samples added annually. These archived samples contain a wealth of genetic information and offer a great potential for discovery and analysis of biomarkers with diagnostic and therapeutic significance.1Lewis 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 (267) Google Scholar,2Shibata D Extraction of DNA from paraffin-embedded tissue for analysis by polymerase chain reaction: new tricks from an old friend.Hum Pathol. 1994; 25: 561-563Abstract Full Text PDF PubMed Scopus (92) Google Scholar Laser capture microdissected (LCM) samples,3Emmert-Buck MR Bonner RF Smith PD Chuaqui RF Zhuang Z Goldstein SR Weiss RA Liotta LA Laser capture microdissection.Science. 1996; 274: 998-1001Crossref PubMed Scopus (2176) Google Scholar tissue print micropeels,4Gaston SM Soares MA Siddiqui MM Vu D Lee JM Goldner DL Brice MJ Shih JC Upton MP Perides G Baptista J Lavin PT Bloch BN Genega EM Rubin MA Lenkinski RE Tissue-print and print-phoresis as platform technologies for the molecular analysis of human surgical specimens: mapping tumor invasion of the prostate capsule.Nat Med. 2005; 11: 95-101Crossref PubMed Scopus (29) Google Scholar and plasma-circulating DNA5Anker P Mulcahy H Chen XQ Stroun M Detection of circulating tumour DNA in the blood (plasma/serum) of cancer patients.Cancer Metastasis Rev. 1999; 18: 65-73Crossref PubMed Scopus (411) Google Scholar are additional sources of valuable clinical material with major potential for discovery and evaluation of cancer biomarkers. However, the quantity and condition of the DNA trapped in such diverse clinical samples has proven to be a significant barrier.1Lewis 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 (267) Google Scholar Knowledge of DNA quality is important to determine the types of techniques that the material can support. For example, the quality of archived specimens is dependent on fixation and storage conditions and can be highly variable between samples.1Lewis 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 (267) Google Scholar,6Koch I Slotta-Huspenina J Hollweck R Anastasov N Hofler H Quintanilla-Martinez L Fend F Real-time quantitative RT-PCR shows variable, assay-dependent sensitivity to formalin fixation: implications for direct comparison of transcript levels in paraffin-embedded tissues.Diagn Mol Pathol. 2006; 15: 149-156Crossref PubMed Scopus (40) Google Scholar In addition, because specimen yield is often a limiting factor in studying nonrenewable clinical samples, the ability to assess DNA quality with a minimal amount of material before investing time and resources for sample analysis is of paramount importance.7Siwoski A Ishkanian A Garnis C Zhang L Rosin M Lam WL An efficient method for the assessment of DNA quality of archival microdissected specimens.Mod Pathol. 2002; 15: 889-892Crossref PubMed Scopus (30) Google Scholar Recent advances in whole genome amplification (WGA)8Dean 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 (1142) Google Scholar9Wang G Maher E Brennan C Chin L Leo C Kaur M Zhu P Rook M Wolfe JL Makrigiorgos GM DNA amplification method tolerant to sample degradation.Genome Res. 2004; 14: 2357-2366Crossref PubMed Scopus (76) Google Scholar10Telenius H Carter NP Bebb CE Nordenskjold M Ponder BA Tunnacliffe A Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer.Genomics. 1992; 13: 718-725Crossref PubMed Scopus (1232) Google Scholar11Zhang L Cui X Schmitt K Hubert R Navidi W Arnheim N Whole genome amplification from a single cell: implications for genetic analysis.Proc Natl Acad Sci USA. 1992; 89: 5847-5851Crossref PubMed Scopus (808) Google Scholar12Makrigiorgos GM Chakrabarti S Zhang Y Kaur M Price BD A PCR-based amplification method retaining the quantitative difference between two complex genomes.Nat Biotechnol. 2002; 20: 936-939Crossref PubMed Scopus (68) Google Scholar13Klein CA Schmidt-Kittler O Schardt JA Pantel K Speicher MR Riethmuller G Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells.Proc Natl Acad Sci USA. 1999; 96: 4494-4499Crossref PubMed Scopus (368) Google Scholar14Hughes S Sellick G Coleman R Langmore J GenomePlex whole genome amplification.in: Hughes S Lasken RS Whole Genome Application. Scion Publishing Limited, Oxford2005: 59-76Google Scholar15Hughes S Arneson N Done S Squire J The use of whole genome amplification in the study of human disease.Prog Biophys Mol Biol. 2005; 88: 173-189Crossref PubMed Scopus (76) Google Scholar have made it possible to gain access to sufficient quantities of DNA from clinical samples to perform a number of downstream applications including real-time quantitative polymerase chain reaction (QRT-PCR), comparative genomic hybridization (CGH), and single nucleotide polymorphism (SNP) microarrays.8Dean 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 (1142) Google Scholar9Wang G Maher E Brennan C Chin L Leo C Kaur M Zhu P Rook M Wolfe JL Makrigiorgos GM DNA amplification method tolerant to sample degradation.Genome Res. 2004; 14: 2357-2366Crossref PubMed Scopus (76) Google Scholar10Telenius H Carter NP Bebb CE Nordenskjold M Ponder BA Tunnacliffe A Degenerate oligonucleotide-primed PCR: general amplification of target DNA by a single degenerate primer.Genomics. 1992; 13: 718-725Crossref PubMed Scopus (1232) Google Scholar11Zhang L Cui X Schmitt K Hubert R Navidi W Arnheim N Whole genome amplification from a single cell: implications for genetic analysis.Proc Natl Acad Sci USA. 1992; 89: 5847-5851Crossref PubMed Scopus (808) Google Scholar12Makrigiorgos GM Chakrabarti S Zhang Y Kaur M Price BD A PCR-based amplification method retaining the quantitative difference between two complex genomes.Nat Biotechnol. 2002; 20: 936-939Crossref PubMed Scopus (68) Google Scholar13Klein CA Schmidt-Kittler O Schardt JA Pantel K Speicher MR Riethmuller G Comparative genomic hybridization, loss of heterozygosity, and DNA sequence analysis of single cells.Proc Natl Acad Sci USA. 1999; 96: 4494-4499Crossref PubMed Scopus (368) Google Scholar14Hughes S Sellick G Coleman R Langmore J GenomePlex whole genome amplification.in: Hughes S Lasken RS Whole Genome Application. Scion Publishing Limited, Oxford2005: 59-76Google Scholar The success of WGA itself also depends on sample quality.9Wang G Maher E Brennan C Chin L Leo C Kaur M Zhu P Rook M Wolfe JL Makrigiorgos GM DNA amplification method tolerant to sample degradation.Genome Res. 2004; 14: 2357-2366Crossref PubMed Scopus (76) Google Scholar,16Wang G Brennan C Rook M Wolfe JL Leo C Chin L Pan H Liu WH Price B Makrigiorgos GM Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples.Nucleic Acids Res. 2004; 32: e76Crossref PubMed Scopus (58) Google Scholar Certain types of WGA such as multiple displacement amplification perform well with fresh or snap-frozen (intact) DNA,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 (217) Google Scholar18Lovmar L Syvanen AC Multiple displacement amplification to create a long-lasting source of DNA for genetic studies.Hum Mutat. 2006; 27: 603-614Crossref PubMed Scopus (84) Google Scholar19Lovmar L Fredriksson M Liljedahl U Sigurdsson S Syvanen A-C Quantitative evaluation by minisequencing and microarrays reveals accurate multiplexed SNP genotyping of whole genome amplified DNA.Nucl Acids Res. 2003; 31: e129Crossref PubMed Scopus (116) Google Scholar but their yield9Wang G Maher E Brennan C Chin L Leo C Kaur M Zhu P Rook M Wolfe JL Makrigiorgos GM DNA amplification method tolerant to sample degradation.Genome Res. 2004; 14: 2357-2366Crossref PubMed Scopus (76) Google Scholar and accuracy20Rook MS Delach SM Deyneko G Worlock A Wolfe JL Whole genome amplification of DNA from laser capture-microdissected tissue for high-throughput single nucleotide polymorphism and short tandem repeat genotyping.Am J Pathol. 2004; 164: 23-33Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar,21Li J Harris L Mamon H Kulke M Liu W Zhu P Makrigiorgos GM Whole genome amplification of plasma-circulating DNA enables expanded screening for allelic imbalance in plasma.J Mol Diagn. 2006; 8: 22-30Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar diminish on DNA fragmentation. WGA methods that can tolerate various degrees of sample degradation have been recently developed.9Wang G Maher E Brennan C Chin L Leo C Kaur M Zhu P Rook M Wolfe JL Makrigiorgos GM DNA amplification method tolerant to sample degradation.Genome Res. 2004; 14: 2357-2366Crossref PubMed Scopus (76) Google Scholar,14Hughes S Sellick G Coleman R Langmore J GenomePlex whole genome amplification.in: Hughes S Lasken RS Whole Genome Application. Scion Publishing Limited, Oxford2005: 59-76Google Scholar,16Wang G Brennan C Rook M Wolfe JL Leo C Chin L Pan H Liu WH Price B Makrigiorgos GM Balanced-PCR amplification allows unbiased identification of genomic copy changes in minute cell and tissue samples.Nucleic Acids Res. 2004; 32: e76Crossref PubMed Scopus (58) Google Scholar For example, restriction and circularization-aided rolling circle amplification (RCA-RCA), which circularizes fragmented DNA and then applies rolling-circle-initiated multiple displacement amplification, can produce WGA of partially degraded DNA from archived genomes sufficient in both fidelity and yield for quantitative PCR and copy number analysis via microarray-based comparative genomic hybridization.9Wang G Maher E Brennan C Chin L Leo C Kaur M Zhu P Rook M Wolfe JL Makrigiorgos GM DNA amplification method tolerant to sample degradation.Genome Res. 2004; 14: 2357-2366Crossref PubMed Scopus (76) Google Scholar,22Makrigiorgos GM Genome amplification tolerant to sample degradation: application to formalin-fixed, paraffin-embedded specimens.in: Hughes S Lasken RS Whole Genome Application. Scion Publishing Limited, Oxford2005: 149-161Google Scholar Similarly, plasma-circulating DNA can be amplified via RCA-RCA and used in expanded genetic screening.21Li J Harris L Mamon H Kulke M Liu W Zhu P Makrigiorgos GM Whole genome amplification of plasma-circulating DNA enables expanded screening for allelic imbalance in plasma.J Mol Diagn. 2006; 8: 22-30Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar However, the investment required for performing such procedures is not justified if there is no guarantee on the suitability of the starting DNA. A simple assay that predicts success of WGA as well as downstream assays for small amounts of starting DNA such as that obtained via LCM, tissue print micropeels,4Gaston SM Soares MA Siddiqui MM Vu D Lee JM Goldner DL Brice MJ Shih JC Upton MP Perides G Baptista J Lavin PT Bloch BN Genega EM Rubin MA Lenkinski RE Tissue-print and print-phoresis as platform technologies for the molecular analysis of human surgical specimens: mapping tumor invasion of the prostate capsule.Nat Med. 2005; 11: 95-101Crossref PubMed Scopus (29) Google Scholar or plasma-circulating DNA would be very useful because it would prevent wasting time, effort, and resources. Ethidium bromide-based agarose gel electrophoresis requires more than 100 ng of DNA to visualize the extended fragmentation associated with archived DNA and does not consistently predict success of PCR from archived samples.23van Beers EH Joosse SA Ligtenberg MJ Fles R Hogervorst FB Verhoef S Nederlof PM A multiplex PCR predictor for aCGH success of FFPE samples.Br J Cancer. 2006; 94: 333-337Crossref PubMed Scopus (194) Google Scholar Methods that use competitive PCR amplification of short-versus-long amplicons have been used for assessing DNA quality and fragmentation.7Siwoski A Ishkanian A Garnis C Zhang L Rosin M Lam WL An efficient method for the assessment of DNA quality of archival microdissected specimens.Mod Pathol. 2002; 15: 889-892Crossref PubMed Scopus (30) Google Scholar,23van Beers EH Joosse SA Ligtenberg MJ Fles R Hogervorst FB Verhoef S Nederlof PM A multiplex PCR predictor for aCGH success of FFPE samples.Br J Cancer. 2006; 94: 333-337Crossref PubMed Scopus (194) Google Scholar24Cawkwell L Quirke P Direct multiplex amplification of DNA from a formalin fixed, paraffin wax embedded tissue section.Mol Pathol. 2000; 53: 51-52Crossref PubMed Scopus (33) Google Scholar25Johnson NA Hamoudi RA Ichimura K Liu L Pearson DM Collins VP Du MQ Application of array CGH on archival formalin-fixed paraffin-embedded tissues including small numbers of microdissected cells.Lab Invest. 2006; 86: 968-978Crossref PubMed Scopus (76) Google Scholar26Lehmann 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 (320) Google Scholar However, most cannot address the requirements posed by the minute starting material obtained via microdissection of tumor samples. Published multiplex PCR predictor assays require 100 ng of starting material,23van Beers EH Joosse SA Ligtenberg MJ Fles R Hogervorst FB Verhoef S Nederlof PM A multiplex PCR predictor for aCGH success of FFPE samples.Br J Cancer. 2006; 94: 333-337Crossref PubMed Scopus (194) Google Scholar but DNA amounts extracted from LCM samples are usually less than 10 to 20 ng.3Emmert-Buck MR Bonner RF Smith PD Chuaqui RF Zhuang Z Goldstein SR Weiss RA Liotta LA Laser capture microdissection.Science. 1996; 274: 998-1001Crossref PubMed Scopus (2176) Google Scholar,20Rook MS Delach SM Deyneko G Worlock A Wolfe JL Whole genome amplification of DNA from laser capture-microdissected tissue for high-throughput single nucleotide polymorphism and short tandem repeat genotyping.Am J Pathol. 2004; 164: 23-33Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar Rapid amplification of polymorphic DNA (RAPD-PCR)-based assays7Siwoski A Ishkanian A Garnis C Zhang L Rosin M Lam WL An efficient method for the assessment of DNA quality of archival microdissected specimens.Mod Pathol. 2002; 15: 889-892Crossref PubMed Scopus (30) Google Scholar use a few nanograms of starting material but examine genomic regions outside housekeeping genes. Because tumor samples are frequently altered because of genomic instability,27Maeda T Jikko A Hiranuma H Fuchihata H Analysis of genomic instability in squamous cell carcinoma of the head and neck using the random amplified polymorphic DNA method.Cancer Lett. 1999; 138: 183-188Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar RAPD-PCR-amplified sequences cannot distinguish between deletion of chromosomal regions and changes in sample quality in cancer samples. We describe a housekeeping gene-based multiplex-PCR approach that, when combined with fluorescence detector-based denaturing high-performance liquid chromatography (dHPLC), possesses the sensitivity required to assess sample quality from 1 ng of genomic DNA after a single multiplex PCR reaction. Alternatively, the method may also be used with a common ethidium bromide-stained agarose gel when accurate quantification is not a major issue. Starting with 1 ng of genomic DNA, a multiplex PCR is performed, and the PCR product is screened via dHPLC or an agarose gel (Figure 1). The use of high-sensitivity fluorescence detection enables easier identification of small amounts of intact DNA exceeding 300 to 400 bases that is instrumental for the success of WGA via RCA-RCA and downstream assays such as quantitative PCR analysis (Figure 1). We report the establishment and validation of this new dHPLC-based approach for accurately assessing DNA quality in archived samples of varying fixation conditions, age, and degradation as well as in minute amounts of LCM, tissue micropeels, and plasma-circulating DNA. Reference human male genomic DNA was purchased from Promega (Madison, WI). High-grade glioblastoma/glioma and colon cancer formalin-fixed, paraffin-embedded (FFPE) specimens (times after fixation/FFPE age: ∼5 to 7 years and ∼10 to 12 years, respectively) were obtained from Medical Oncology, Dana Farber Cancer Institute and Brigham and Women's Hospital. Plasma samples from radiation therapy cancer patients were obtained from Radiation Oncology, Brigham and Women's–Dana Farber Cancer Center. The collection and use of unidentifiable human specimens for genetic analysis was approved by the institutional review board. Genomic DNA from snap-frozen tissues was extracted and purified with the DNAeasy kit (Qiagen, Valencia, CA). Genomic DNA was prepared from FFPE glioma and colon specimens using a modified method. In brief, ∼25 mg of tissue per sample was deparaffinized by treatment with mixed xylenes (1.2 ml; vortexed, centrifuged 3 minutes at room temperature, removal of xylene, and repeated one to two times until clear), and xylenes were removed by addition of 100% ethanol (1.2 ml; vortexed, centrifuged 3 minutes at room temperature, removal of ethanol, and repeated one to two times until clear). After vaporization of ethanol for 10 minutes at 37°C, samples were washed in phosphate-buffered saline (PBS) (1.2 ml; vortexed, centrifuged 3 minutes at room temperature, removal of PBS). Tissue was placed in 360 μl of lysis buffer (Qiagen) and 40 μl of proteinase K (Qiagen) and rotated at 55°C for 24 to 72 hours as needed for full digestion. Subsequent DNA purification was performed using the DNAeasy kit, adjusting buffer and extraction volumes for the volume of lysis buffer used. DNA extracted from FFPE specimens was initially evaluated by gel electrophoresis of 0.75 μg of DNA in a 1% agarose gel. To extract plasma-circulating DNA, whole blood was centrifuged at 2000 × g for 15 to 30 minutes within 2 hours of collection, and plasma was carefully collected from the top of the supernatant, as we described earlier.21Li J Harris L Mamon H Kulke M Liu W Zhu P Makrigiorgos GM Whole genome amplification of plasma-circulating DNA enables expanded screening for allelic imbalance in plasma.J Mol Diagn. 2006; 8: 22-30Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar Plasma-circulating DNA was purified from plasma with QIAamp MinElute virus spin kit (Qiagen) and quantified using the PicoGreen method (Molecular Probes, Eugene, OR) and via real-time PCR for the GAPDH gene as was described previously.21Li J Harris L Mamon H Kulke M Liu W Zhu P Makrigiorgos GM Whole genome amplification of plasma-circulating DNA enables expanded screening for allelic imbalance in plasma.J Mol Diagn. 2006; 8: 22-30Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar To obtain DNA from snap-frozen tumor tissue and from the corresponding FFPE specimens, snap-frozen human breast cancer tissue was dissected into equal-sized fragments. Some fragments were retained frozen, whereas others were fixed in neutral buffered formalin (10%) for 24 hours. After processing, all fragments were sectioned at 50-μm thickness and processed for DNA extraction. To obtain DNA from tumor tissue samples kept at room temperature for varying waiting times before fixation and for varying times within formalin, we used xenograph tumors from the human MCAS ovarian cell line grown in vivo. MCAS cells (0.5 × 106) mixed with Matrigel (BD Biosciences, San Diego, CA) were injected subcutaneously in the right flank of an 8-week-old female nude mouse (nu/nu; Charles River Laboratories, Wilmington, MA). After 4 weeks the subcutaneous tumor was harvested and dissected into 10 equally sized fragments. One fragment was immediately snap-frozen and retained at −80°C. Three tumor fragments were fixed immediately in 10% neutral buffered formalin for 2, 24, and 48 hours, respectively, before embedding in paraffin. A second and third group of tumor fragments were retained at room temperature for 1 hour and 5 hours, respectively, in PBS before fixation and paraffin embedding in an identical manner. After processing each block was sectioned at a thickness of 50 μm. For LCM samples, snap-frozen prostate tissues were cut on membrane slides (Molecular Devices Corp., formerly Arcturus Bioscience, Sunnyvale, CA) at 7-μm thickness. The slides were stained with HistoGene LCM frozen section staining kit (Molecular Devices) and then processed via LCM (Veritas Instrument; Arcturus Bioscience Inc.). Alternatively, manual microdissection was performed on the same tissue sections. The captured samples were treated for DNA extraction via overnight proteinase K digestion at 56°C followed by extraction with phenol/chloroform and precipitation with 0.5 mol/L ammonium acetate, glycogen, and 2 vol of 100% ethanol. The DNA was then eluted in Tris 10 mmol/L, pH 8.0, and quantified by Quant-iT PicoGreen double-stranded DNA reagent and kits (Molecular Probes-Invitrogen). Prostate cancer tissue print micropeels were obtained from fresh tissue slices obtained from radical prostatectomy specimens at the time of surgery, as we reported previously.4Gaston SM Soares MA Siddiqui MM Vu D Lee JM Goldner DL Brice MJ Shih JC Upton MP Perides G Baptista J Lavin PT Bloch BN Genega EM Rubin MA Lenkinski RE Tissue-print and print-phoresis as platform technologies for the molecular analysis of human surgical specimens: mapping tumor invasion of the prostate capsule.Nat Med. 2005; 11: 95-101Crossref PubMed Scopus (29) Google Scholar Prostate tissue prints were snap-frozen immediately on collection and stored at −80°C before processing; each tissue print was sliced into a set of 5 × 5-mm tiles for biomarker extraction. Print collection and processing was performed in an oriented manner (with fiduciary markers) so that FFPE samples from corresponding tumor sites could be identified for DNA analysis. Biological material was extracted from the tissue print nitrocellulose by immersing the frozen tissue print tile in a buffer containing guanidine-isothiocyanate (Qiagen buffer RLT) and applying mechanical agitation. After removal of insoluble debris, separate DNA and RNA fractions were purified from the tissue print extract using the Qiagen AllPrep DNA/RNA kit. Each 5 × 5-mm prostate tissue print tile produced ∼1 to 2 μg of DNA. The corresponding FFPE prostate tumor samples were less than 6 months old at the time they were extracted for DNA following the procedure described above. Multiplex PCR to assess sample quality was performed using ∼1 ng of purified DNA from fresh or archived DNA, or alternatively using 1 μl of plasma-circulating DNA. The forward and reverse primers used to co-amplify genomic segments of 105, 236, 299, and 411 bp, respectively, from the GAPDH gene (GenBank ID J04038) were as follows: 5′-GGCTGAGAACGGGAAGCTTG-3′ and 5′-ATCCTAGTTGCCTCCCCAAA-3′ (105 bp); 5′-CGGGTCTTTGCAGTCGTATG-3′ and 5′-GCGAAAGGAAAGAAAGCGTC-3′ (236 bp); 5′-AGGTGAGACATTCTTGCTGG-3′ and 5′-TCCACTAACCAGTCAGCGTC-3′ (299 bp); and 5′-TGAATGGGCAGCCGTTAGGAAAGC-3′ and 5′-AGACACCCAATCCTCCCGGTGACA-3′ (411 bp). A 25-μl PCR reaction was set up that included a final concentration of 0.24, 0.60, 1.2, and 0.30 μmol/L of the four primers 105, 236, 299, and 411 bp, respectively, 300 μmol/L dNTP, 1.5 mmol/L MgCl2, 1× PCR buffer, and 0.25 μl of GoTaq polymerase 9 (Promega) and the interrogated genomic DNA. Alternatively, JumpStart Taq polymerase from Sigma, St. Louis, MO, was used for some experiments. Thermocycling was performed in a MiniOpticon machine (Bio-Rad, Hercules, CA) using the following program: 94°C for 2 minutes, followed with 40 cycles at 94°C for 30 seconds then 56°C for 30 seconds and 72°C for 1 minute. Then a final extension at 72°C for 3 minutes was performed. PCR products were analyzed either by ethidium bromide-stained 1% agarose gel electrophoresis or via HPLC chromatography on a WAVE dHPLC system (Transgenomics, Inc., Omaha, NE). The WAVE system is equipped with a fluorescence detector and with two 96-well autosamplers that enable high-throughput analysis of PCR products. The use of the high-sensitivity fluorescence detector in conjunction with SYBR Green I dye infusion enables detection and quantification of picogram amounts of PCR amplicons. The dHPLC was run at nondenaturing temperatures (50°C) for analysis of multiplex PCR products. Restriction and circularization-aided rolling circle amplification (RCA-RCA) was used for WGA of genomic DNA extracted from snap-frozen and archived clinical samples. The published RCA-RCA protocol9Wang G Maher E Brennan C Chin L Leo C Kaur M Zhu P Rook M Wolfe JL Makrigiorgos GM DNA amplification method tolerant to sample degradation.Genome Res. 2004; 14: 2357-2366Crossref PubMed Scopus (76) Google Scholar was used with minor modifications. In brief, 20 to 50 ng of genomic DNA was digested with 5 U of NlaIII restriction endonuclease (0.5 μl from a 10 U/μl stock, catalog no. R0125S; New England Biolabs, Beverly, MA) in 10 μl of 1× T4 DNA ligase buffer (catalog no. M0202M; New England Biolabs) for 2 hours at 37°C. The enzyme was then inactivated via heating at 65°C for 20 minutes. The digested DNA was circularized by adding 0.5 μl of T4 DNA ligase (200 U), 0.5 μl of T4 ligase buffer, and 4 μl of water to the digested DNA solution and incubating at room temperature for 2 hours. T4 DNA ligase was then inactivated by heating at 65°C for 10 minutes. Linear DNA was then eliminated with 1.2 μl of Lamda exonuclease and 0.3 μl of exonuclease I (New England Biolabs) in a volume of 25 μl at 37°C for 1 hour. The balance of the volume to 25 μl was made up in Lamda exonuclease buffer. The circularized ligation product was then used in a standard multiple displacement WGA reaction using the GenomiPh
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