- Technical Note
- Open Access
Laser capture microdissection (LCM) and whole genome amplification (WGA) of DNA from normal breast tissue --- optimization for genome wide array analyses
© Hedenfalk et al; licensee BioMed Central Ltd. 2011
- Received: 9 September 2010
- Accepted: 18 March 2011
- Published: 18 March 2011
Laser capture microdissection (LCM) can be applied to tissues where cells of interest are distinguishable from surrounding cell populations. Here, we have optimized LCM for fresh frozen normal breast tissue where large amounts of fat can cause problems during microdissection. Since the amount of DNA needed for genome wide analyses, such as single nucleotide polymorphism (SNP) arrays, is often greater than what can be obtained from the dissected tissue, we have compared three different whole genome amplification (WGA) kits for amplification of DNA from LCM material. In addition, the genome wide profiling methods commonly used today require extremely high DNA quality compared to PCR based techniques and DNA quality is thus critical for successful downstream analyses.
We found that by using FrameSlides without glass backing for LCM and treating the slides with acetone after staining, the problems caused by excessive fat could be significantly decreased. The amount of DNA obtained after extraction from LCM tissue was not sufficient for direct SNP array analysis in our material. However, the two WGA kits based on Phi29 polymerase technology (Repli-g® (Qiagen) and GenomiPhi (GE Healthcare)) gave relatively long amplification products, and amplified DNA from Repli-g® gave call rates in the subsequent SNP analysis close to those from non-amplified DNA. Furthermore, the quality of the input DNA for WGA was found to be essential for successful SNP array results and initial DNA fragmentation problems could be reduced by switching from a regular halogen lamp to a VIS-LED lamp during LCM.
LCM must be optimized to work satisfactorily in difficult tissues. We describe a work flow for fresh frozen normal breast tissue where fat is inclined to cause problems if sample treatment is not adapted to this tissue. We also show that the Phi29-based Repli-g® WGA kit (Qiagen) is a feasible approach to amplify DNA of high quality prior to genome wide analyses such as SNP profiling.
- Normal Breast Tissue
- Laser Capture Microdissection
- Single Nucleotide Polymorphism Array
- Whole Genome Amplification
- Multiple Displacement Amplification
Laser capture microdissection (LCM) is a widely used method for isolation of defined cell populations from heterogeneous tissue sections. The method allows selection of unmixed starting material for DNA, RNA or protein extraction for further downstream molecular analyses [1–3]. However, LCM needs to be optimized depending on tissue type, for example normal breast tissue contains more lipids than breast tumor tissue. The PALM MicroBeam system (Carl Zeiss MicroImaging, Jena, Germany) is one of several commercially available LCM systems. This system utilizes a UV laser to cut around the selected cells and a pulse from the same laser to catapult the selected specimen into a collection device, e.g. an AdhesiveCap microcentrifuge tube (Carl Zeiss MicroImaging). In order to catapult larger tissue structures with a single laser pulse, slides with thin polyethylene membranes are used. There are two types of slides available; MembraneSlides, which are regular glass slides covered by the membrane and FrameSlides where the membrane is only supported by a metal frame.
The amount of DNA yielded by LCM can be sufficient for PCR based analyses, but is often insufficient for genome-wide applications such as high density single nucleotide polymorphism (SNP) genotyping arrays. Whole genome amplification (WGA) provides a possibility to amplify a small amount of high quality DNA and there are several WGA methods available. Many WGA kits on the market today employ the multiple displacement amplification (MDA) technology (e.g. GenomiPhi (GE Healthcare Life Sciences, Uppsala, Sweden) and Repli-g® (Qiagen, Hilden, Germany)). This Phi29 DNA polymerase-based technique has in many studies been found to provide the most balanced genome amplification to date [4–7]. The concordance between non-amplified and MDA amplified DNA in SNP arrays has been found to be higher than 98% [8–10] and a majority of studies report MDA WGA to be the method of choice for SNP array analyses [5, 11, 12]. However, the key to accurate WGA is high quality input DNA and the DNA quality after LCM is difficult to investigate since the small amount of DNA obtained after microdissection restricts the methods available for quality assurance.
In this study, we have developed a protocol for LCM of fresh frozen normal breast tissue to enable microdissection of this challenging, fat rich tissue. We have also tested three different, commercially available, WGA kits to obtain sufficient amounts of high quality DNA for high-density SNP array analysis.
Tissue preparation and laser capture microdissection (LCM)
DNA extraction and whole genome amplification (WGA)
DNA was extracted directly after dissection using the QIAmp DNA Micro kit (Qiagen) according to their specific protocol for extraction of genomic DNA from LCM tissues. A few modifications were made to the protocol: Buffer ATL and proteinase K were mixed before addition to the AdhesiveCap and no vortexing of the tubes was performed before 4 hours incubation at 56ºC in an up-side-down position. DNA concentrations were measured on the Qubit™ quantification platform using the Quant-iT™ high sensitivity assay for double stranded DNA (dsDNA) (Invitrogen, Carlsbad, CA). The DNA yield was approximately 30 ng DNA per mm2 of microdissected tissue.
WGA using three different commercial kits on DNA extracted from microdissected tissue and from blood
Recommended input (μg)
Expected output (μg)
Repli-g (Qiagen) ®
GenomiPhi (GE Healthcare)
BioScore™ (Enzo Life Sciences)
Call rates for SNP arrays
Direct ampl. Repli-g ®
SNP analyses were run on Infinium Human Omni 1 M arrays (Illumina, San Diego, CA) at the Swegene Centre for Integrative Biology at Lund University (SCIBLU) genomics facility (Lund University, Sweden). Data analyses were performed in Illumina's GenomeStudio® Genotyping Module. The SNP array call rates are listed in Table 2 and show that amplification products from both blood and tissue with the Repli-g® kit (Qiagen) give call rates comparable to what was obtained with non-amplified blood and tissue. Non-amplified DNA, however, showed less noise in the SNP profiles. GenomiPhi and BioScore™ amplified DNA from blood caused more noise in the SNP analysis and also displayed clearly lower call rates than non-amplified DNA from blood. These kits were therefore not further applied to DNA from LCM tissue. Interestingly, the direct-amplified (Repli-g®, Qiagen) LCM tissue without previous DNA extraction gave extremely poor call rates, and the high noise level suggests that this amplification method is clearly not suited before genome wide analyses.
We have developed a protocol for LCM of fresh frozen normal breast tissue, which is a demanding tissue to work with and we believe the method to be valid for other lipid-rich tissues as well. A switch to FrameSlides and an added acetone wash were necessary for LCM to work adequately. When using FrameSlides, however, it was obvious that some tissue was dropped and could be found on the objective below the stage. This loss, together with other pieces that were not successfully catapulted into the cap, was estimated to approximately 10% and is in line with observations made by other groups . Interestingly, other groups working with breast tissue have not described problems with LCM. However, much of the previous work has been performed on paraffin embedded tissue [15, 16] where all lipids have been removed during the embedding process. For frozen sections, most work has been performed on breast tumor tissue [2, 17], which contains considerably less adipose cells than normal breast tissue. To our knowledge, in the few studies where normal fresh frozen breast tissue has been studied, LCM systems by Arcturus Engineering have been used [18–22]. Possibly, these systems are less sensitive to contaminating lipids.
We performed a thorough quality control of DNA obtained from LCM tissue after recognizing that a lack of long fragments in the input to WGA did not result in unsuccessful amplification but was only observable as low SNP array call rates. As displayed in the gel image in Figure 2a, the majority of the LCM DNA fragments were shorter than 4 kb. We found that the original halogen lamp in the LCM microscope was causing DNA fragmentation during microdissection, probably due to heat production. After changing to a VIS-LED lamp, clearly higher DNA quality was obtained (Figure 2b). We suspect that poor DNA quality is often a problem in the analysis of DNA obtained from LCM, but that this is rarely discovered since the small amount of DNA limits the options for quality control of long fragment DNA. Most likely, the DNA quality after LCM is sufficient for most PCR-based assays and possibly for other less sensitive analyses. However, we found that for SNP array analyses the quality of the input DNA is crucial for a successful outcome after WGA.
The authors wish to thank Srinivas Veerla for help with data analysis. This work was supported by grants from the Swedish Cancer Society, the Swedish Research Council, the Crafoord Foundation, the King Gustav V Jubilee Fund, the Gunnar Nilsson Cancer Foundation, the Berta Kamprad Cancer Foundation, the University Hospital of Lund Research Foundation and Governmental Funding of Clinical Research within National Health Service.
- Bowen NJ, Walker LD, Matyunina LV, Logani S, Totten KA, Benigno BB, McDonald JF: Gene expression profiling supports the hypothesis that human ovarian surface epithelia are multipotent and capable of serving as ovarian cancer initiating cells. BMC Med Genomics. 2009, 2: 71-10.1186/1755-8794-2-71.PubMedPubMed CentralView ArticleGoogle Scholar
- Umar A, Kang H, Timmermans AM, Look MP, Meijer-van Gelder ME, den Bakker MA, Jaitly N, Martens JW, Luider TM, Foekens JA, Pasa-Tolić L: Identification of a putative protein profile associated with tamoxifen therapy resistance in breast cancer. Mol Cell Proteomics. 2009, 8: 1278-94. 10.1074/mcp.M800493-MCP200.PubMedPubMed CentralView ArticleGoogle Scholar
- Gagnon JF, Sanschagrin F, Jacob S, Tremblay AA, Provencher L, Robert J, Morin C, Diorio C: Quantitative DNA methylation analysis of laser capture microdissected formalin-fixed and paraffin-embedded tissues. Exp Mol Pathol. 2010, 88: 184-9. 10.1016/j.yexmp.2009.09.020.PubMedView ArticleGoogle Scholar
- Dean 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. Pros Nat Acad Sci USA. 2002, 99: 5261-66. 10.1073/pnas.082089499.View ArticleGoogle Scholar
- Lovmar L, Syvänen AC: Multiple displacement amplification to create a long-lasting source of DNA for genetic studies. Hum Mut. 2006, 27: 603-614. 10.1002/humu.20341.PubMedView ArticleGoogle Scholar
- Panelli S, Damiani G, Espen L, Micheli G, Sgaramella V: Towards the analysis of the genomes of single cells: Further characterization of the multiple displacement amplification. Gene. 2006, 372: 1-7. 10.1016/j.gene.2006.01.032.PubMedView ArticleGoogle Scholar
- Lasken RS: Genomic amplification by the multiple displacement amplification (MDA) method. Biochem Soc Trans. 2009, 37: 450-453. 10.1042/BST0370450.PubMedView ArticleGoogle Scholar
- Barker DL, Hansen MS, Faruqi AF, Giannola D, Irsula OR, Lasken RS, Latterich M, Makarov V, Oliphant A, Pinter JH, Shen R, Sleptsova I, Qiehler W, Lai E: Two methods of whole-genome amplification enable accurate genotyping across a 2320-SNP linkage panel. Genome Res. 2004, 14: 901-907. 10.1101/gr.1949704.PubMedPubMed CentralView ArticleGoogle Scholar
- Paez JG, Lin M, Beroukhim R, Lee JC, Zhao X, Richter DJ, Gabriel S, Herman P, Sasaki H, Altshuler D, Li C, Meyerson M, Sellers WR: Genome coverage and sequence fidelity of phi29 polymerase-based multiple strand displacement whole genome amplification. Nucleic Acids Res. 2004, 32: e71-10.1093/nar/gnh069.PubMedPubMed CentralView ArticleGoogle Scholar
- Xing J, Watkins WS, Zhang Y, Witherspoon DJ, Jorde LB: High fidelity of whole-genome amplified DNA on high-density single nucleotide polymorphism arrays. Genomics. 2008, 92: 452-456. 10.1016/j.ygeno.2008.08.007.PubMedPubMed CentralView ArticleGoogle Scholar
- Jasmine F, Ahsan H, Andrulis IL, John EM, Chang-Claude J, Kibriya MG: Whole-genome amplification enables accurate genotyping for microarray-based high-density single nucleotide polymorphism array. Cancer Epidemiol Biomarkers Prev. 2008, 17: 3499-3508. 10.1158/1055-9965.EPI-08-0482.PubMedPubMed CentralView ArticleGoogle Scholar
- Gunderson KL, Steemers FJ, Lee G, Mendoza LG, Chee MS: A genome-wide scalable SNP genotyping assay using microarray technology. Nat Gen. 2005, 37: 549-554. 10.1038/ng1547.View ArticleGoogle Scholar
- Rennstam K, Ringberg A, Cunliffe HE, Olsson H, Landberg G, Hedenfalk I: Genomic alterations in histopathologically normal breast tissue from BRCA1 mutation carriers may be caused by BRCA1 haploinsufficiency. Genes Chromosomes Cancer. 2010, 49 (1): 78-90. 10.1002/gcc.20723. Erratum in: Genes Chromosomes Cancer. 2010, 49(8):760-1.PubMedView ArticleGoogle Scholar
- Wang WZ, Oeschger FM, Lee S, Molnár Z: High quality RNA from multiple brain regions simultaneously acquired by laser capture microdissection. BMC Mol Biol. 2009, 10: 69-10.1186/1471-2199-10-69.PubMedPubMed CentralView ArticleGoogle Scholar
- Balogh GA, Heulings R, Mailo D, Wang R, Li YS, Hardy R, Russo J: Methodological approach to study the genomic profile of the human breast. Int J Oncol. 2007, 31 (2): 253-260.PubMedGoogle Scholar
- Green AR, Young P, Krivinskas S, Rakha EA, Claire Paish E, Powe DG, Ellis IO: The expression of ERalpha, ERbeta and PR in lobular carcinoma in situ of the breast determined using laser microdissection and real-time PCR. Histopathology. 2009, 54 (4): 419-427. 10.1111/j.1365-2559.2009.03233.x.PubMedView ArticleGoogle Scholar
- Yang F, Foekens JA, Yu J, Sieuwerts AM, Timmermans M, Klijn JG, Atkins D, Wang Y, Jiang Y: Laser microdissection and microarray analysis of breast tumors reveal ER-alpha related genes and pathways. Oncogene. 2006, 25 (9): 1413-1419. 10.1038/sj.onc.1209165.PubMedView ArticleGoogle Scholar
- King C, Guo N, Frampton GM, Gerry NP, Lenburg ME, Rosenberg CL: Reliability and reproducibility of gene expression measurements using amplified RNA from laser-microdissected primary breast tissue with oligonucleotide arrays. J Mol Diagn. 2005, 7 (1): 57-64. 10.1016/S1525-1578(10)60009-8.PubMedPubMed CentralView ArticleGoogle Scholar
- Tripathi A, King C, de la Morenas A, Perry VK, Burke B, Antoine GA, Hirsch EF, Kavanah M, Mendez J, Stone M, Gerry NP, Lenburg ME, Rosenberg CL: Gene expression abnormalities in histologically normal breast epithelium of breast cancer patients. Int J Cancer. 2008, 122 (7): 1557-66. 10.1002/ijc.23267.PubMedView ArticleGoogle Scholar
- Frolova N, Edmonds MD, Bodenstine TM, Seitz R, Johnson MR, Feng R, Welch DR, Frost AR: A shift from nuclear to cytoplasmic breast cancer metastasis suppressor 1 expression is associated with highly proliferative estrogen receptor-negative breast cancers. Tumour Biol. 2009, 30 (3): 148-159. 10.1159/000228908.PubMedPubMed CentralView ArticleGoogle Scholar
- Ong KR, Sims AH, Harvie M, Chapman M, Dunn WB, Broadhurst D, Goodacre R, Wilson M, Thomas N, Clarke RB, Howell A: Biomarkers of dietary energy restriction in women at increased risk of breast cancer. Cancer Prev Res (Phila Pa). 2009, 2 (8): 720-31. 10.1158/1940-6207.CAPR-09-0008.View ArticleGoogle Scholar
- Graham K, de Las Morenas A, Tripathi A, King C, Kavanah M, Mendez J, Stone M, Slama J, Miller M, Antoine G, Willers H, Sebastiani P, Rosenberg CL: Gene expression in histologically normal epithelium from breast cancer patients and from cancer-free prophylactic mastectomy patients shares a similar profile. Br J Cancer. 2010, 102 (8): 1284-93. 10.1038/sj.bjc.6605576.PubMedPubMed CentralView ArticleGoogle Scholar
- Ling J, Zhuang G, Tazon-Vega B, Zhang C, Cao B, Rosenwaks Z, Xu K: Evaluation of genome coverage and fidelity of multiple displacement amplification from single cells by SNP array. Mol Hum Reprod. 2009, 15: 739-747. 10.1093/molehr/gap066.PubMedPubMed CentralView ArticleGoogle Scholar
- Cardoso J, Molenaar L, de Menezes RX, Rosenberg C, Morreau H, Moslein G, Fodde R, Boer JM: Genomic profiling by DNA amplification of laser capture microdissected tissues and array CGH. Nucleic Acids Res. 2004, 32: e146-10.1093/nar/gnh142.PubMedPubMed CentralView ArticleGoogle Scholar
- Rook MS, Delach SM, Deyneko G, Worlock A, Wolfe JL: Whole genome amplification of DNA from laser capture-mictodissected tissue for high-throughput single nucleotide polymorphism and short tandem repeat genotyping. Am J Pathol. 2004, 164: 23-33. 10.1016/S0002-9440(10)63092-1.PubMedPubMed CentralView ArticleGoogle Scholar
- Hughes S, Lim G, Beheshti B, Bayani J, Marrano P, Huang A, Squire JA: Use of whole genome amplification and comparative genomic hybridization to detect chromosomal copy number alterations in cell line material and tumour tissue. Cytogenet Genome Res. 2004, 105: 18-24. 10.1159/000078004.PubMedView ArticleGoogle Scholar
- Frumkin D, Wasserstrom A, Itzkovitz S, Harmelin A, Rechavi G, Shapiro E: Amplification of multiple genomic loci from single cells isolated by laser micro-dissection of tissues. BMC Biotech. 2008, 8: 17-10.1186/1472-6750-8-17.View ArticleGoogle Scholar
- Arriola E, Lambros MBK, Jones C, Dexter T, Mackay A, Tan DSP, Tamber N, Fenwick K, Ashworth A, Dowsett M, Reis-Filho JS: Evaluation of Phi29-based whole-genome amplification for microarray-based comparative genomic hybridization. Lab Invest. 2007, 87: 75-83. 10.1038/labinvest.3700495.PubMedView ArticleGoogle Scholar
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