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.
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.
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