Whole Genome Amplification of DNA from Laser Capture-Microdissected Tissue for High-Throughput Single Nucleotide Polymorphism and Short Tandem Repeat Genotyping
2004; Elsevier BV; Volume: 164; Issue: 1 Linguagem: Inglês
10.1016/s0002-9440(10)63092-1
ISSN1525-2191
AutoresMartha Rook, Scott Delach, Galina Deyneko, Andrew Worlock, Jia Liu Wolfe,
Tópico(s)DNA Repair Mechanisms
ResumoGenome-wide screening of genetic alterations between normal and cancer cells, as well as among subgroups of tumors, is important for establishing molecular mechanism and classification of cancer. Gene silencing through loss of heterozygosity is widely observed in cancer cells and detectable by analyzing allelic loss of single nucleotide polymorphism and/or short tandem repeat markers. To use minute quantities of DNA that are available through laser capture microdissection (LCM) of cancer cells, a whole genome amplification method that maintains locus and allele balance is essential. We have successfully used a ø29 polymerase-based isothermal whole genome amplification method to amplify LCM DNA using a proteinase K lysis procedure coupled with a pooling strategy. Through single nucleotide polymorphism and short tandem repeat genotype analysis we demonstrate that using pooled DNA from two or three separate amplification reactions significantly reduces any allele bias introduced during amplification. This strategy is especially effective when using small quantities of source DNA. Although a convenient alkaline lysis DNA extraction procedure provided satisfactory results from using 1500 to 3000 LCM cells, proteinase K digestion was superior for lower cell numbers. Accurate genotyping is achieved with as few as 100 cells when both proteinase K extraction and pooling are applied. Genome-wide screening of genetic alterations between normal and cancer cells, as well as among subgroups of tumors, is important for establishing molecular mechanism and classification of cancer. Gene silencing through loss of heterozygosity is widely observed in cancer cells and detectable by analyzing allelic loss of single nucleotide polymorphism and/or short tandem repeat markers. To use minute quantities of DNA that are available through laser capture microdissection (LCM) of cancer cells, a whole genome amplification method that maintains locus and allele balance is essential. We have successfully used a ø29 polymerase-based isothermal whole genome amplification method to amplify LCM DNA using a proteinase K lysis procedure coupled with a pooling strategy. Through single nucleotide polymorphism and short tandem repeat genotype analysis we demonstrate that using pooled DNA from two or three separate amplification reactions significantly reduces any allele bias introduced during amplification. This strategy is especially effective when using small quantities of source DNA. Although a convenient alkaline lysis DNA extraction procedure provided satisfactory results from using 1500 to 3000 LCM cells, proteinase K digestion was superior for lower cell numbers. Accurate genotyping is achieved with as few as 100 cells when both proteinase K extraction and pooling are applied. The revelation that cancer is a genomic disease along with the availability of draft human genome sequence have motivated the development of high-throughput technologies that can detect genetic alterations between normal and cancer cells, as well as differences among subgroups of tumors.1Weber BL Cancer genomics.Cancer Cell. 2002; 1: 37-47Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar Recently, gene expression profiling research on molecular classification of cancer highlighted the advantage of a genome-wide perspective on genetic variations.2Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM Lymphoma/Leukemia Molecular Profiling Project: The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3274) Google Scholar, 3van 't Veer LJ Dai H van de Vijver MJ He YD Hart AA Mao M Peterse HL van der Kooy K Marton MJ Witteveen AT Schreiber GJ Kerkhoven RM Roberts C Linsley PS Bernards R Friend SH Gene expression profiling predicts clinical outcome of breast cancer.Nature. 2002; 415: 530-536Crossref PubMed Scopus (7968) Google Scholar, 4Ramaswamy S Ross KN Lander ES Golub TR A molecular signature of metastasis in primary solid tumors.Nat Genet. 2003; 33: 49-54Crossref PubMed Scopus (2056) Google Scholar The altered mRNA levels in cancer genomes are often related to gene amplification of growth factor receptors, or the loss of functional tumor suppressor genes through homozygous deletion or loss of heterozygosity.5Thiagalingam S Foy RL Cheng KH Lee HJ Thiagalingam A Ponte JF Loss of heterozygosity as a predictor to map tumor suppressor genes in cancer: molecular basis of its occurrence.Curr Opin Oncol. 2002; 14: 65-72Crossref PubMed Scopus (92) Google Scholar, 6Quon KC Berns A Haplo-insufficiency? Let me count the ways.Genes Dev. 2001; 15: 2917-2921Crossref PubMed Scopus (85) Google Scholar Detection of such gene copy number changes has been achieved by comparative genomic hybridization analysis.7Forozan F Karhu R Kononen J Kallioniemi A Kallioniemi OP Genome screening by comparative genomic hybridization.Trends Genet. 1997; 13: 405-409Abstract Full Text PDF PubMed Scopus (248) Google Scholar However, because loss of heterozygosity may be accompanied by chromosome multiplicity or duplication of a dysfunctional allele, the most reliable approach to identify loss of heterozygosity is through detection of locus-specific genotype loss using a panel of single nucleotide polymorphism (SNP) markers and short tandem repeat (STR) or microsatellite markers. Additionally, STR genotyping may reveal the presence of microsatellite instability in cancer cells,8Andrew SE Peters AC DNA instability and human disease.Am J Pharmacogenomics. 2001; 1: 21-28Crossref PubMed Scopus (14) Google Scholar while SNP genotyping may identify potential cancer risk and drug effects that are attributable to SNPs.9Roses AD Genome-based pharmacogenetics and the pharmaceutical industry.Nat Rev Drug Discov. 2002; 1: 541-549Crossref PubMed Scopus (136) Google Scholar To ensure accurate data interpretation, it is desirable to use laser capture microdissection (LCM) to separate cancer cells from surrounding normal cells in tumor lesions.10Emmert-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 To perform genotyping assays in a genome-wide manner, a whole genome amplification (WGA) method that amplifies DNA from a small number of LCM cells while maintaining locus and allele balance is necessary. To date, several WGA methods, including degenerated oligonucleotide primed-polymerase chain reaction (DOP-PCR),11Cheung 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 (226) Google Scholar, 12Harada T Okita K Shiraishi K Kusano N Furuya T Oga A Kawauchi S Kondoh S Sasaki K Detection of genetic alterations in pancreatic cancers by comparative genomic hybridization coupled with tissue microdissection and degenerate oligonucleotide primed polymerase chain reaction.Oncology. 2002; 62: 251-258Crossref PubMed Scopus (32) Google Scholar, 13Grant SF Steinlicht S Nentwich U Kern R Burwinkel B Tolle R SNP genotyping on a genome-wide amplified DOP-PCR template.Nucleic Acids Res. 2002; 30: e125Crossref PubMed Scopus (31) Google Scholar primer extension preamplification (PEP),14Zhang 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 (807) Google Scholar, 15Dietmaier W Hartmann A Wallinger S Heinmoller E Kerner T Endl E Jauch KW Hofstadter F Ruschoff J Multiple mutation analyses in single tumor cells with improved whole genome amplification.Am J Pathol. 1999; 154: 83-95Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar, 16Wang VW Bell DA Berkowitz RS Mok SC Whole genome amplification and high-throughput allelotyping identified five distinct deletion regions on chromosomes 5 and 6 in microdissected early-stage ovarian tumors.Cancer Res. 2001; 61: 4169-4174PubMed Google Scholar and linker-adaptor ligation-based SCOMP,17Klein 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 Scholar, 18Stoecklein NH Erbersdobler A Schmidt-Kittler O Diebold J Schardt JA Izbicki JR Klein CA SCOMP is superior to degenerated oligonucleotide primed-polymerase chain reaction for global amplification of minute amounts of DNA from microdissected archival tissue samples.Am J Pathol. 2002; 161: 43-51Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar have been used to amplify DNA extracted from LCM cells. Each of these methods has limitations in terms of incomplete genomic coverage, low extent of amplification, or complex experimental manipulations. Moreover, these methods have been applied primarily for comparative genomic hybridization analysis that has somewhat lenient requirements for gene coverage and allele bias. To the best of our knowledge, there has been no previous report on using WGA products for SNP genotyping using a TaqMan platform, which is known for its accuracy, ease of operation, and amenability to automation.19De La Vega FM Dailey D Ziegle J Williams J Madden D Gilbert DA New generation pharmacogenomic tools: a SNP linkage disequilibrium map, validated SNP assay resource, and high-throughput instrumentation system for large-scale genetic studies.Biotechniques. 2002; : S48-S54PubMed Google Scholar Recently, an isothermal WGA method using the strand-displacing ø29 polymerase was developed and successfully applied to amplify DNA from cell culture and blood.20Dean 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 Scholar This method, termed "whole genome multiple strand displacement amplification (MDA)," demonstrated a high-amplification potential (up to 104-fold) and excellent loci representation (less than threefold bias).20Dean 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 Scholar To evaluate this method for amplification of DNA from LCM cells, we used Amersham's ø29 polymerase-based GenomiPhi kit (Amersham Biosciences, Piscataway, NJ) and assessed the quality of amplified products relative to unamplified DNA using SNP- and STR-genotyping assays. Using LCM cells collected from cancer and normal samples of colon and prostate, we found that DNA isolated from LCM cells generated more pronounced allelic bias than cell culture DNA, and that this bias seemed to inversely correlate with template quantity. Decreased allele imbalance was observed when proteinase K digestion was used to isolate DNA instead of the standard alkaline lysis method, probably because of a more complete digestion of cellular proteins and better release of DNA.15Dietmaier W Hartmann A Wallinger S Heinmoller E Kerner T Endl E Jauch KW Hofstadter F Ruschoff J Multiple mutation analyses in single tumor cells with improved whole genome amplification.Am J Pathol. 1999; 154: 83-95Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar In addition, we found that accurate genotype calling rates were much higher when amplified DNA products were pooled from two or three separate amplification reactions before analysis, and that this effect was especially dramatic when less cells were used. With pooling, greater than 95% of SNP and STR accurate genotype calling rates were achieved from alkaline lysis of ∼1500 cells, whereas proteinase K digestion rendered effective performance with as few as 100 cells. Isolated human DNA samples from six cell lines representing two sets of trios (mother, father, and offspring) were obtained from the Coriell Cell Repositories (Camden, NJ). Amplification was performed using the GenomiPhi WGA kit (Amersham Biosciences, Piscataway, NJ) according to kit instructions. Briefly, 5 ng of DNA (1 μl) was added to 9 μl of sample buffer and the mixture was heat-denatured at 95°C for 3 minutes. After cooling on ice for 5 minutes, 10 μl of reaction mix (9 μl of reaction buffer plus 1 μl of ø29 polymerase) was added and the resulting mixture was incubated at 30°C for 16 hours. The polymerase was heat-inactivated at 65°C for 10 minutes. Each sample was diluted twofold using 1× TE (10 mmol/L Tris-HCl pH 8.0, 1 mmol/LEDTA) (pH 8.0) and its DNA concentration measured using the Hoechst dye assay (Bio-Rad, Hercules, CA). Typically 3 to 6 μg of amplified DNA was obtained from 5 ng of input DNA in a 20-μl WGA reaction. Normal and matching primary prostate cancer samples from eight patients were obtained from Clinomics (Pittsfield, MA). Normal and cancer colon samples of two individuals were obtained from the Cooperative Human Tissue Network. Fresh-frozen samples were embedded in OCT, sections 8-μm thick were cut in cryostat at − 23°C and mounted on slides covered by polyethylene membrane (PEN slides; C. Zeiss, Thornwood, NY). The resulting tissue slides were stored at −80°C until microdissection. Each slide was rinsed with sterile water for 1 minute, 70% ethanol for 1 minute, and stained in Mayer's hematoxylin (Sigma Chemical Co., St. Louis, MO) for 30 seconds. After twice rinsing with deionized water, the slide was treated with staining bluing reagent (Harleco, EM Science, Gibbstown, NJ) for 1 minute, 70% ethanol for 1 minute, 95% ethanol for 1 minute, 1% Eosin Y in alcohol (Harleco) for 20 seconds, 95% ethanol for 1 minute, twice more with fresh 95% ethanol for 1 minute, and 100% ethanol for 3 minutes. After allowed to air-dry for 5 minutes with the airflow turned on, the stained slides were microdissected within 2 hours. A pathologist (GD) performed LCM using a P.A.L.M. Robot-Microbeam System (Oberkochen, Germany) following the manufacturer's recommendations. First, the cell density on a standard hematoxylin and eosin-stained coverslipped slide was determined by counting the number of cells in an area of 1000 μm2Rosenwald A Wright G Chan WC Connors JM Campo E Fisher RI Gascoyne RD Muller-Hermelink HK Smeland EB Giltnane JM Hurt EM Zhao H Averett L Yang L Wilson WH Jaffe ES Simon R Klausner RD Powell J Duffey PL Longo DL Greiner TC Weisenburger DD Sanger WG Dave BJ Lynch JC Vose J Armitage JO Montserrat E Lopez-Guillermo A Grogan TM Miller TP LeBlanc M Ott G Kvaloy S Delabie J Holte H Krajci P Stokke T Staudt LM Lymphoma/Leukemia Molecular Profiling Project: The use of molecular profiling to predict survival after chemotherapy for diffuse large-B-cell lymphoma.N Engl J Med. 2002; 346: 1937-1947Crossref PubMed Scopus (3274) Google Scholar (×400 magnification) using software provided by the manufacturer of the P.A.L.M. This result was then used to calculate how many square microns are needed for a desired number of cells to be collected. Typical LCM images before and after microdissection are shown in Figure 1A (×100 to ×200 magnification). Immediately after the capture of cells, the cap containing the cells was placed on top of its matching Eppendorf tube and centrifuged for 1 minute at 14,000 rpm (16,000 × g). The cap/tube was snap-frozen in a dry ice/ethanol bath, and the collection of LCM cells contained within was hereafter referred to as a LCM cell cap or a cap. The cells were stored at −80°C for up to 2 months before DNA extraction. DNA was extracted from LCM cells using either an alkaline lysis or a proteinase K lysis protocol. When using the alkaline lysis method, 5 μl of 1× phosphate-buffered saline was added to the cap containing LCM cells, followed by 5 μl of lysis buffer (400 mmol/L KOH, 100 mmol/L dithiothreitol, 10 mmol/L ethylenediaminetetraacetic acid). The mixture was pipetted multiple times to facilitate the collection of all cells from the cap. The 10-μl suspension was transferred to its matching Eppendorf tube and vortexed for 1 minute. After incubation on ice for 10 minutes, the solution was neutralized by addition of 5 μl of neutralization buffer containing 0.4 mol/L HCl and 0.6 mol/L Tris-HCl, pH 7.5. When using the proteinase K lysis protocol, 15 μl of a proteinase K buffer containing 0.4 mg/ml of proteinase K from Qiagen (Valencia, CA), 1× TE (pH 8.0), and 1% Tween-20 was added to a LCM cell cap, and mixed with the cells by pipetting multiple times. The 15-μl suspension was transferred to its matching Eppendorf tube, vortexed for 1 minute, and incubated at 55°C for 3 hours. The amount of DNA from a typical 3000 cell LCM cap was measured by quantitative PCR analysis using primers targeting PGK1 and TFRC genes. Five μl of lysed DNA was added to assay reagents to obtain a 70-μl PCR mix containing 1× ABI TaqMan master mix, 4 μmol/L 5′ primer, 4 μmol/L 3′ primer, and 2 μmol/L probe. For each sample, triplicates of 20 μl of PCR reactions were run and a standard curve used to determine the amount of DNA containing the probed locus as described in the quantitative PCR and relative copy number section (see below). The calculated concentrations of the PGK1 and TFRC loci in μg/μl were averaged and used to calculate the total μg of isolated DNA per LCM cap. MDA was performed using the GenomiPhi WGA kit (Amersham) according to kit instructions with some modifications for LCM samples. Five μl of lysed DNA from LCM cells was added to 20 μl of sample buffer, heated at 95°C for 3 minutes, and then cooled on ice for 5 minutes. Twenty-five μl of reaction mix (22.5 μl of reaction buffer plus 2.5 μl of ø29 polymerase) was added and the resulting samples were incubated at 30°C for 16 hours. After heat denaturation at 65°C for 10 minutes, each sample was diluted twofold using 1× TE (pH 8.0) and its DNA concentration was measured using the Hoechst dye assay (Bio-Rad). Typically 12 to 16 μg of amplified DNA product was obtained from a 50-μl reaction mixture. TaqMan genotyping was performed using an ABI PRISM 7900HT sequence detection system following the manufacturer's instructions (Applied Biosystems, Foster City, CA). Assays targeting 12 different genetic loci were designed through ABI's Assay-by-Design service and used for genotyping analysis. Duplicate reactions were run for each assay with a 384-well plate, with control DNA samples run on the same plate. PCR was performed with 20 ng of input DNA, 1× ABI TaqMan master mix, 1 μmol/L of each primer, and 0.25 μmol/L of each probe. Fluorescence intensities (arbitrary units) of the two probes were plotted and genotype calling was performed using predefined calling parameters. Calling parameters for each genotype were determined beforehand based on the clustering of a panel of 101 genomic DNA samples obtained from Coriell. Briefly, we first defined the plot regions that contained allele 1, allele 2, or heterozygous genotypes using lines with lower and upper intensities and slopes (eg, as shown in Figure 1, Figure 3, Figure 4). Genotyping data points that fell within these defined regions were called, data points that fell outside these regions, and those below intensity values as defined by no template controls were considered no calls. Miscalls were assigned to data points that were in contradiction to a genotype call made by either an unamplified sample of the same tissue or a consensus call based on all of the replicate assays performed on the same amplified tissue sample (pooled, unpooled, different cell numbers, and so forth). The consensus calls based on amplified material were always in agreement with the accurate genotype calls from unamplified samples when both sets of data were available. Calling rates (Table 1, Table 3) were the percentage of calls (accurate calls plus miscalls where applicable) out of all assays performed. Accurate calling rates (Table 2) were the percentage of correct genotype calls out of all of the genotyping assays performed.Figure 4A: Effect of pooling WGA products from a 300 or a 100 LCM prostate cell cap on TaqMan SNP-genotyping assay for A/G36177 of the CYP3A5 gene: 24 WGA products were assayed in replicates for each cell number to afford 96 unpooled data points; 8 individual WGA products were assayed in replicates for each cell number to afford 32 pooled data points. No calls are highlighted by open circles and miscalls are highlighted by filled circles. Shown on axes are arbitrary fluorescence units. B: Effect of pooling WGA products from a 300 colon cell cap on a STR-genotyping assay using a 2-bp repeat marker D13S173.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Table 1Effect of LCM Cell Number and Lysis Method on Genotyping Performance of Whole Genome Amplified DNA using Φ29 Polymerase-Based GenomiPhi ReagentsSNP analysisSTR analysisPerformance dataNo. of loci assayedPerformance dataNo. of colon cells/capLysis methodNo. of amp. rxn./capNo. of loci assayed% Calling% Cap-to-cap concordance% Replicate concordance% Calling% Cap-to-cap concordance% Replicate concordance3000Alkaline2496.9100.095.81099.0100.098.3Proteinase K2497.9100.093.810100.0100.0100.01500Alkaline2992.090.097.51095.697.595.0Proteinase K2690.592.9100.0998.8100.096.7750Alkaline21279.671.488.91084.270.096.7Proteinase K2895.994.197.21095.098.397.5300Alkaline21249.033.395.81068.180.083.8Proteinase K2883.088.297.01096.795.091.3100Alkaline30nd*Not determined.nd*Not determined.nd*Not determined.0nd*Not determined.nd*Not determined.nd*Not determined.Proteinase K3775.0100.097.6997.991.791.7* Not determined. Open table in a new tab Table 3WGA Methods for Amplification of LCM DNA: A Comparison Based on References and Our ResultsSNP analysisSTR analysisPerformance dataPerformance dataWGA methodNo. of LCM cells/capAmp. yield as no. of assaysPlatform% Calling% WrongTotal no. of assays runPlatform% Calling% WrongTotal no. of assays runDOP-PCR*Based on reference 15.100¶FACS-sorted cultured cells.nsNot specified.ndNot determined.ndNot determined.ndNot determined.ndNot determined.Slap gel/EtBr85.0020PEP*Based on reference 15.25010 SNP or 30 STRRFLP: Slap gel/EtBr94.7019Slap gel/EtBr98.8082PEP†Based on reference 16.5000150 STRndNot determined.ndNot determined.ndNot determined.ndNot determined.ABI/florescencendNot determined.ndNot determined.nsNot specified.SCOMP‡Based on reference 18.48∥Cells from 3-month-old formalin-fixed tissues.32 STRndNot determined.ndNot determined.ndNot determined.ndNot determined.Slap gel/SYBR green94.45.618Pooled MDA§Summary based on our experiments. The LCM cell numbers were presented as a range to reflect potential ∼twofold underestimation.100–200**Lysed cells were divided into three portions and amplified separately then pooled.2000 SNP or 4000 STRTaqMan/florescence87.10124ABI/florescence99.10232Pooled MDA§Summary based on our experiments. The LCM cell numbers were presented as a range to reflect potential ∼twofold underestimation.300–600Lysed cells were divided into three portions, two of which were amplified separately then pooled (the third portion was stored for future use); the effective cell number was 200–400.2000 SNP or 4000 STRTaqMan/florescence93.90164ABI/florescence97.50.6362Not amplified§Summary based on our experiments. The LCM cell numbers were presented as a range to reflect potential ∼twofold underestimation.3000–600050 SNP or 50 STRTaqMan/florescence58.9056ABI/florescence97.5040All data was obtained using fresh-frozen tissues except where indicated for which comparable data on fresh-frozen tissues was not available.* Based on reference 15Dietmaier W Hartmann A Wallinger S Heinmoller E Kerner T Endl E Jauch KW Hofstadter F Ruschoff J Multiple mutation analyses in single tumor cells with improved whole genome amplification.Am J Pathol. 1999; 154: 83-95Abstract Full Text Full Text PDF PubMed Scopus (210) Google Scholar.† Based on reference 16Wang VW Bell DA Berkowitz RS Mok SC Whole genome amplification and high-throughput allelotyping identified five distinct deletion regions on chromosomes 5 and 6 in microdissected early-stage ovarian tumors.Cancer Res. 2001; 61: 4169-4174PubMed Google Scholar.‡ Based on reference 18Stoecklein NH Erbersdobler A Schmidt-Kittler O Diebold J Schardt JA Izbicki JR Klein CA SCOMP is superior to degenerated oligonucleotide primed-polymerase chain reaction for global amplification of minute amounts of DNA from microdissected archival tissue samples.Am J Pathol. 2002; 161: 43-51Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar.§ Summary based on our experiments. The LCM cell numbers were presented as a range to reflect potential ∼twofold underestimation.¶ FACS-sorted cultured cells.∥ Cells from 3-month-old formalin-fixed tissues.** Lysed cells were divided into three portions and amplified separately then pooled.†† Lysed cells were divided into three portions, two of which were amplified separately then pooled (the third portion was stored for future use); the effective cell number was 200–400.‡‡ Not specified.§§ Not determined. Open table in a new tab Table 2SNP and STR Genotyping Results from Pooling Two or Three Amplification Reactions Starting with Proteinase K-Lysed Colon or Prostate CellsSNP analysisSTR analysisTissue no. of patientsNo. of LCM cells/capAvg. amp. foldNo. of WGA rxn. pooledNo. of loci assayedTotal no. of assays% Accurate callingNo. of loci assayedTotal no. of assays% Accurate callingProstate 215004.5 × 103364895.81080100.0 47501.0 × 104369694.81016095.0 43002.3 × 104369699.01016096.9 41006.4 × 104369689.610160100.0Colon 215004.5 × 103264892.9972100.0 27501.0 × 10421296100.0108090.6 23002.3 × 1042129694.1108097.5 21006.4 × 104375681.597297.2Each cell number represents duplicate caps. Each cap of cells was lysed and the mixture divided into three portions, two or three of which were separately amplified then pooled (see Figure 2). The average amplification fold was calculated from a large number of WGA reactions, including those not shown in this table. All assays were run in replicates, and percentage accurate calling is the percentage of accurate genotype calls for all assays performed. Open table in a new tab All data was obtained using fresh-frozen tissues except where indicated for which comparable data on fresh-frozen tissues was not available. Each cell number represents duplicate caps. Each cap of cells was lysed and the mixture divided into three portions, two or three of which were separately amplified then pooled (see Figure 2). The average amplification fold was calculated from a large number of WGA reactions, including those not shown in this table. All assays were run in replicates, and percentage accurate calling is the percentage of accurate genotype calls for all assays performed. STR genotyping was performed using Applied Biosystems Linkage Mapping Set of fluorescence-labeled primer pairs. A total of 45 different assays targeting various loci were used for this study. For each assay, replicate reactions were run for every DNA sample. PCR reactions were run with a 7.5-μl total volume containing 10 ng DNA, 1× ABI True Allele master mix, and 0.2 μl ABI primers. Reactions were incubated at 95°C for 12 minutes, cycled 15 times at 95°C for 15 seconds, 55°C for 15 seconds, 72°C for 30 seconds, and cycled an additional 25 times at 89°C for 15 seconds, 55°C for 15 seconds, 72°C for 30 seconds, with a final extension at 72°C for 10 minutes. PCR products were pooled (for multiplexing) and run on an ABI PRISM 3700 DNA analyzer. Data analysis was performed using ABI PRISM Genotyper Software Version 3.7. Peak height ratios between 0.4 and 6 were called as heterozygotes, peak height ratios between 0.1 and 0.4 and that between 6 and 10 were defined as no calls. Accurate calling rates were the percentage of correct genotype calls of all of the genotyping experiments. The relative copy numbers of four genes, GAPD, TBP, TFRC, and PPIA, were determined for WGA products from three different origins. The first set of samples (×4) were prepared by pooling three WGA products from 1500 LCM colon cell caps. The second set of samples (×4) were pooled from three WGA products originating from 100 LCM colon cell caps. The final set of samples (×6) were from WGA reactions using 5 ng of genomic DNA isolated from six different cell lines as templates. The concentration of each amplified DNA product was determined by a Picogreen fluorescence assay (Molecular Probes, Eugene, OR), and a stock solution at 3 ng/μl was prepared for each sample. Fifteen ng (5 μl) of each amplified DNA sample was added to assay reagents to obtain a 70-μl PCR mix containing 1× ABI TaqMan master mix, 4 μmol/L 5′ primer, 4 μmol/L 3′ primer, and 2 μmol/L probe. For each sample, triplicates of 20 μl of PCR were run. All PCRs were run in a 384-well plate using an ABI PRISM 7900HT detection system. The cycling prog
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