MMI Bibliography III

 

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Laser Microdissection for Transcriptomics

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molecular machines industries -bibliography vol 3 no 1 2010 issue on laser microdissection for transcriptomics

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»lead the way to micromanipulation« contents in this issue this issue is dedicated to all researchers who wish to study gene expression in individual cells and tissue areals after laser microdissection it provides tips and tricks for the set up of representative work flow`s starting from fresh frozen or ffpe material and features helpful publications in this field customer report single or small subsets of cells reveal clean-cut transcriptome profiles by eric cabuy meng msc phd single cell genomics friedrich miescher institute for biomedical research basel switzerland using laser microdissection and transcriptional profiling to identify the molecular constituents of taste buds by bryan d moyer ph.d former associate director ion channel biology senomyx inc san diego ca usa mmi report designing a work flow for rna based analysis using laser microdissected materials by the mmi team mmi ag featured publications references mmi-bibliography online www.molecular-machines.com/news the lmd-bibliograhy is published by molecular machines industries.com issn 1662-8535 editor dr antje plaschke-schluetter e-mail schluetter@molecular-machines.com editorial board norbert brill andre imhof prof stefan seeger concept layout kathrin plaschke ­ grafik design ­ hamburg the mmi caplift technology is patented by molecular machines industries mmi is a registered trademark www.molecular-machines.com mmi-bibliography 2

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»lead the way together with you« editorial the isolation of intact rna from tiny amounts of starting material is the most critical step for sound gene expression analysis based on microarrays microarrays have long been established for the generation of gene expression profiles that reflect differences between normal and diseased states differently expressed genes may have the potential to serve as new targets in drug discovery but getting a proper sample from bedsite or the operation suit to bench is a monumental task demanding an all-out effort and a strictly harmonized work flow between all people involved the rising demand for new biomarkers as drug targets that could eventually be used to fight life threatening diseases like cancer and neurological disorders fostered the development of new products and protocols based on laser microdissection laser microdissection is a well accepted technology and is used to specifically cut out diseased versus non diseased cells from heterogeneous biopsy material the aim of this third issue of the mmi bibliography is to show recent advancements in the protocol development for transcriptomics after laser microdissection it introduces outstanding approaches and turn key steps for the implementation of such work flows the report from eric cabuy who heads the single cell genomics facility at the friedrich miescher institute for biomedical research in basel switzerland is focussed on gene expression profiling of motoneurons from fresh frozen sections of the mouse brain this work was recently published in nature neuroscience in his report bryan moyer introduces the strategy that allowed senomyx inc to generate the first comprehensive taste bud gene expression library in primates and rodents this work is based on highthrough put laser microdissection of fresh frozen primate and rodent tongue blocks coupled to whole genome transcriptional profiling after isothermal amplification the contribution from our team entitled designing a work flow rna based analysis using laser microdissected material .for microarray or quantitative real-time pcr analysis highlights the individual steps for work flow implementation and refers to actual publications on protocol development and optimization the whole mmi team hopes that you enjoy this issue in case you feel that you would like to contribute to future issues please contact us we value your opinion and input which will help us to further strengthen our technical developments based on your needs watch for the seminar announcements on our web-page and the scheduled meetings and trainings our next user-meeting takes place in october 2010 check our web-site for the exact dates program and speakers we look forward to »welcome you to our upcoming user-meeting in october 2010« dr antje plaschke-schluetter mmi ag zuerich www.molecular-machines.com mmi-bibliography 3

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»lead the way to micromanipulation« c u s to merreports single or small subsets of cells reveal `clean-cut transcriptome profiles ever since we are studying the molecular biology of organisms one of the primary efforts is to identify new types of cells and then to elucidate their meaning in molecular biology experiments proteins and nucleic acids are measured as average values in large populations of cells this may lead to the wrong conclusions on the assumption that individual cells differ from each other the vast complexity we see at the nano-scale between individual cells would reveal differences at the single cell level an obvious statement for those studying the physical properties of cells [1 as information is lost by averaging signals it is necessary to study individual or very small subsets of cells in order to determine mechanistic details thirteen years ago zhao already argued that each cell in a tissue is an individual acting in concert with its neighbors [2 the overall behavior of the tissue may therefore be an emergent property of the organization of and interaction between individual cells this hypothesis may have profound consequences for cell biology in september 2005 i joined the friedrich miescher institute fmi in basel switzerland where i was given the task to establish a laboratory in single cell genomics scg scg at the fmi broadly refers to the molecular biology of individual and small subsets of cells that share similar characteristics or functions the facility s main role is to manage laser-dissection microscopy-based projects provide advice on experimental design as well as teaching and centrally deals with the isolation of single cells and down-stream workflows affymetrix microarrays 3 expression gene and tiling are currently the principal way of transcriptome profiling due to the nature of dealing with different projects the workflows for handling small numbers of cells are characterized by different parameters and design with regard to cell source type numbers preparation staining microarrays etc because of this any form of semi-automation cannot be applied at the moment some of these projects may take 2­4 years and therefore the procedural consistency is very important life cells in suspension are isolated individually by hand-controlled micromanipulaoverview of the mmi laser capture microscope set-up at fmi in basel erik cabuy meng msc phd single cell genomics friedrich miescher institute for biomedical research basel switzerland tion or by fluorescence-activated cell sorting facs facs offers a last resort because mechanical dissociation of the tissue and the time needed to stain and process samples contribute to potential changes in gene expression for the majority of projects single cells are isolated with a laser capture microscope lcm we have opted for the lcm from mmi for its superb laser cutting capability short pulse duration high repetition rate its design contamination-free sandwich technology and its userfriendly operation which is the best adapted to our institute s environment of different temporary users for extracting rna cryo-preserved tissues are superior these are cooled down in isopentane before being stored at minus 80 degrees we cryo-cut sections at a specific thickness depending on cell size and place one section at a time on the pet-membrane immediately followed by a single dehydration step we sub the genechip mouse genome 430 2.0 array from affymetrix used in our study sequently proceed to lcm to capture and lyse cells as quickly as possible this is essential for analyzing single cells in order to snap freeze their physiological states the www.molecular-machines.com mmi-bibliography 4

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»lead the way to advanced transcriptomics« c u s to merreports next step which is equally important is to purify rna while maintaining its integrity isolation of single cells in action my experiments on single cells neurons oocytes have shown that glass fiber filters are superior to other means of rna extraction e.g oligo dt-coated magnetic beads which are not very practical in semi-high throughput quality control is performed on amplified crna/cdna using the agilent rna 6000 pico/nano bioanalyzer procedure rna can be amplified from minute samples in different ways by using ivtor pcr-based amplification techniques the method of choice depends on whether microarrays hybridize with rna ssdna or dsdna despite all efforts in protocol development a linear amplification which involves a reverse transcription of mrna by using a poly-t primer linked to a t7 promoter is still the method that i prefer for frozen brain embbeded in tissue-tek on a cryostat ready for sectioning microarray profiling the limitation of capturing only polya tailed mrna gives us at least the advantage of amplifying specifically rna to a large extent one of the projects that we have been working on is a study of amyotrophic lateral sclerosis als a neurodegenerative disorder characterized by loss of motor neurons resulting in progressive paralysis the idea for this study was to longitudinally analyze gene changes in 10 motor neurons per sample in a mouse model of als at the time mid 2005 it was generally considered that amplifying rna from such low numbers of cells for gene expression analysis on affymetrix microarrays was not feasible in order to assess the technical contributions to any observed expres a single dehydrated brain tissue section mounted on top of a membrane-slide sion differences i first tested different ivt-based amplification techniques on 10,000 gradually down to 400 laser-dissected liver cells and microarray data revealed various correlation values in a second experiment rna was taken from lower numbers down to a single neuron using one of the amplification methods largely equal to the eberwine protocol to improve detection level reliability efficiency and speed of workflows gene level signals and quality controls were obtained from genedata s refiner software once the workflow was established i immediately embarked on several projects e.g one in studying als [3 motor neurons were identified by using fluorescently-labeled dextran beads as a tracer injected into muscles 25­30 sections representing approximately 10 motor neurons were micro-dissected and collected per tube i then preformed microarray experiments in batches of maximum 4 times three replicates using 430 2.0 arrays normally for an experiment like this the mean raw expression values are highly reproducible and the average present calls are about 50 the als study clearly demonstrates the feasibility of using the mmi lcm to perfectly capture single cells consistently during the course of a project that may last for several years view onto a membrane slide before and after capturing of single cells references [1 phillips r kondev j theriot j physical biology of the cell 2009 by garland science taylor francis group llc [2 zhao j cell individuality a basic multicellular phenomenon and its role in the pathogenesis of disease medical hypotheses 1995 445 400-2 [3 saxena s cabuy e caroni p a role for motoneuron subtype-selective er stress in disease manifestations of fals mice nature neuroscience 2009 12:627-636 www.molecular-machines.com mmi-bibliography 5 experimental results of microarray data representing the gene expression profiles of two replicates containing each 10 laser-dissected neurons.

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»lead the way to micromanipulation« c u s to merreports identify the molecular constituents of taste buds by using laser microdissection and transcriptional profiling sensory organs are comprised of a heterogeneous collection of cell types including specialized sensory cells support cells nerve fibers blood vessels epithelial cells and connective tissue collection and analysis of pure populations of sensory cells is important to further understand the molecular processes and pathways active in the traditional senses of vision hearing gustation olfaction and mechanosensation as well as the non-traditional senses of balance temperature and pain gustation the sense of taste is mediated by thousands of sparsely distributed taste buds localized on the tongue surface and oral cavity each taste bud is comprised of 50-100 cells that are hard-wired to detect one of the five known taste qualities sweet bitter umami the taste of monosodium glutamate sour and salty [1 taste buds represent less than 1 of the tongue surface therefore a major challenge in taste research is the collection of sufficient quantities of pure taste buds for experimentation historically this has been accomplished by proteolytic dissociation of gustatory tissue followed by manual isolation of taste buds [2 this approach is effective but drawbacks include low throughput damage to taste cells as well as compromised rna integrity if proteolysis is not carefully monitored and contamination of taste buds with surrounding lingual epithelial cells and connective tissue to comprehensively identify the genes processes and pathways active in gestation in the peripheral nervous system senomyx devised an approach to collect and analyze gene expression in pure preparations of taste buds our approach involved laser microdissection of taste buds followed by transcriptional profiling using a combination of microarray and rt-pcr technologies taste buds and surrounding non-gustatory lingual epithelium are collected to identify genes specifically or preferentially expressed in taste cells laser microdissection is a proven effective tool to rapidly isolate pure populations of specific cells from structurally diverse tissues while microarrays and end-point/qpcr allow genome wide or focused interrogation of rna expression patterns respectively fresh frozen primate and rodent tongue blocks are sectioned and stained prior to laser microdissection using the mmi cell cut instrument 10 m sections are prepared corresponding to the thickness of a single taste bud cell a short post-mortem interval less than 5 minutes rnase-free working conditions rapid workflow from cryosectioning to laser microdissection 1-2 hours and staining protocols that minimize tissue hydration such as the ambion lcm staining kit using solutions that are at least 50 anhydrous are crucial to maximize rna integrity we have optimized laser microdissection throughput and can routinely collect over 10,000 taste bud cells in a single day over 1,000 individual taste buds rna is isolated and linearly amplified using one or two step methodologies single primer isothermal amplification using both random and oligo-dt based reverse transcription generates over 5 ug of amplified cdna from 1,000 collected taste bud cells over 100 individual taste buds www.molecular-machines.com mmi-bibliography 6 bryan d moyer ph.d associate director ion channel biology senomyx inc san diego ca usa figure 1 section from front of tongue fungiform papilla with a single taste bud encircled in black and highlighted with red arrow note that the taste bud comprises only a small fraction of the section scale bar is 20 m.

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»lead the way to advanced transcriptomics« c u s to merreports this yield is sufficient for a microarray experiment using either affymetrix exon arrays that query transcript expression at the whole genome level or 3 expression arrays and reproducibly results in 30-50 present calls for a qpcr reaction utilizing 10 ng amplified cdna 500 different genes of interest can be interrogated from a single preparation using optimized staining methods rna purification techniques and amplification protocols high quality cdna is routinely generated from primate and rodent laser microdissected taste buds [3,4 using this approach senomyx recently generated the first comprehensive taste bud gene expression database in primates encompassing over 2,300 genes [4 over 95 of these genes were not previously known to be expressed in taste buds novel pathways and processes active in gustation were elucidated including prevalent roles for both the immune and endocrine systems as well as developmental pathways highlighting stem cell maturation working from this database g protein-coupled receptors and ion channels were identified that are candidates for mediating tastant recognition signal transduction and information coding to the nervous system in addition using laser microdissection coupled with pcr profiling ion channels expressed in mouse taste buds were evaluated and specific voltage-gated sodium channels that form the foundation of taste cell action potentials and information transfer to nerve fibers were identilingual epithelium expression level 10x fied [3 laser microdissection of sensory organs coupled with transcriptional profiling is an attractive approach to comprehensively identify the genes and pathways active in sensory cells future endeavors using labeled sensory cell populations and single cell genomics will further elucidate sensory cell function in normal and diseased states as well as in wild-type and knockout model systems scatter plot showing expression of over 47.000 transcripts in primate taste buds and lingual epithelial cells from microarray experiments each dot corresponds to a single transcript red and orange dots identify taste bud-associated genes taste bud expression level 10x collection of a single taste bud using mmi cell cut laser microdissection system figure 2 a tip of fungiform papilla before laser microdissection b same section after removal of taste bud c isolated taste bud used for molecular analysis scale bar is 20 m from [4 a b c collection of non-gustatory lingual epithelium figure 3 a b references [1 chandrashekar j hoon ma ryba nj zuker cs 2006 the receptors and cells for mammalian taste nature 444 288-294 c a lingual epithelium before laser microdissection b same section after removal of lingual epithelial cell regions c isolated lingual epithelial cells used for molecular analysis scale bar is 20 m from [4 [2 behe p desimone ja avenet p lindemann b 1990 membrane currents in taste cells of the rat fungiform papilla evidence for two types of ca currents and inhibition of k currents by saccharin j gen physiol 96 1061-1084 [3 gao n lu m echeverri f laita b kalabat d et al 2009 voltage-gated sodium channels in taste bud cells bmc neurosci 10 20 [4 hevezi p moyer bd lu m gao n white e et al 2009 genome-wide analysis of gene expression in primate taste buds reveals links to diverse processes plos one 4 e6395 www.molecular-machines.com mmi-bibliography 7

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»lead the way to micromanipulation« mmi reports designing a work flow mmi team members rna based analysis using laser microdissected material lee petruk · miles bellamy · caroline stevens · ludmilla merker · antje plaschke-schluetter the combined application of laser microdissection and rna based gene expression experiments like qpcr or microarray studies has greatly increased during the last couple of years and has been proven many times in publications take a closer look at our reference list a recent publication by harrel et al clearly demonstrates that contaminating cells alter gene signatures when comparing profiles obtained from whole organ preparations versus laser microdissected tissue areas1 success using laser microdissected material can be as easy as using whole tissue sections as long as the proper precautions and considerations are taken2 here we outline a typical rna based lmd experiment and detail the steps that matter for the isolation of intact rna a typical gene expression study can be broken down into the following steps · sample preparation securing the sample from the donor and preparing it this covers either freezing in embedding medium for fresh frozen material or fixation and paraffin embedding in formalin fixed paraffin embedded work flow`s lee petruk national sales manager mmi inc u.s miles bellamy application scientist consultant mmi inc u.s · cell identification sectioning and staining the sample for cell identification · laser micro dissection adjust the lmd instrument to specifically enrich cell populations · extraction purification quality control lysis of microdissected cells and rna purification · amplification amplifying the rna after reverse transcription to maximize the amount available · labeling attaching fluorescent labels to the pcr step for visualization and · molecular analysis of microarray data or qrt-pcr results after hybridization to a gene chip each of these steps can be important and can mean success or failure of the experiment while many different sample types fixation methods and preparation methods can be used for lmd the preferred method is to use fresh frozen material frozen in an oct block for gene expression profiling ffpe material can also be used for rna analysis but there are additional challenges related to the crosstip linking of nucleic acids and proteins during the fixation process current clinical praxis involves fixing of patient material in formalin and embedding the samples in paraffin blocks these blocks are stable over time and represent a huge source of biological indid you know that the illumina dasl ­ assay was developed with mmi cellcut microdissected material see reference li et al caroline stevens marketing mmi ag zurich ludmila merker service technician mmi ag zurich 1 harrel c.j dye w.w harvell e.m.d sartorius a.c horwitz b.k 2008 contaminating cells alter gene signatures in whole organ versus laser capture microdissected tumors a comparison of experimental breast cancers and their lymph node metastases clin exp metastasis 2008 25 1 81-8 2 erickson s.h albert s.p gillespie w.j canales-rodriguez j linehan m.w pinto a.p chuaqui f.r emmert-buck r.m 2009 quantitative rt-pct gene expression analysis of laser microdissected tissue samples nat protoc.2009 4 902-922 www.molecular-machines.com mmi-bibliography 8

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»lead the way to optimized work flow`s « mmi reports formation a number of new developments have made mrna profiling from ffpe tissue a reality and specific ffpe-optimized dna microarray platforms have been developed it turned out that many scientists analyze small interfering rna`s from ffpe material due to their small size of only about 20 to 25 nucleotides sirna can easily be extracted and is not subject to further degradation approved commercial kits are available to fulfill this task even for sample material that was proctip essed according to clinical routine protocols3 this article is particalternative freezing methods ularly focusing on the preparation and quality assessment of rna isopentane cooled over liquid nitrogen isopentane cooled from lasermicrodissected fresh frozen material for the latest develwith dry ice preferred opments in the field of microarray analysis from laser microdissected ffpe material we refer to the special report by tanney kennedy as well as to the short technical report by linton et al and the publication by werner et al fresh frozen sample preparation a cryomold with oct embedded tissue floating on an isopentane cooling bath sample preparation sample preparation starts when the tissue is removed from the patient or animal inherent rnases dnases and proteases will immediately become active starting to degrade their substrates the goal in sample preparation is to preserve all available rna by minimizing the amount of time between tissue removal and freezing at -80c or in liquid nitrogen or by transferring the tissue intip to formalin for fixation in the course of a ffpe protocol the im take care plementation of tissue removal and processing protocols needs snap freezing in liquid to be quality controlled by a series of rna extractions and evalunitrogen induces ice crystals ations using a bioanalyzer or nanodrop instrument ­ see also under rna extraction and quality control the success of laser microdissection experiments strongly follows the rule garbage in garbage out time is well spent on optimizing the sample preparation procedure after successful removal and preservation steps the sample is then cut onto membrane slides using a cryostat for frozen material or a microtome for ffpe material sections are cut at a thickness between 5 and 10 microns and frozen ready to use cell identification and laser microdissection staining is not completely necessary for all experiments ­ some scientist`s identify the cells or tissue areas of interest without any staining by viewing them under a basic fix stain protocol · 30 75 etoh · 30 h2o · 30 60 stain quick fix hydrolize sample to accept aqueous stain optimize stain concentration to fit this time frame removes excess stain gradual dehydration to maintain morphology · 30 95 etoh · 30 100 etoh · 5 xylene · 5 air dry gradual dehydration to maintain morphology sample is completely dehydrated removes etoh removes the xylene or 100 etoh · 30 h2o · 30 75 etoh can be omitted but sample may absorb some water from the atmosphere due to hydrophilic nature of 100 etoh frozen brain embbeded in tissue-tek on a cryostat ready for sectioning 3 hlubek f schuster c budczies j kirchner t 2010 robust microrna expression profiling of specific cells in complex archival tissue stained by immunohistochemistry abstract at aacr 2010 and paper in submitted www.molecular-machines.com mmi-bibliography 9

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»lead the way to micromanipulation« mmi reports phase contrast ­ but it is very common to identify the cells of interest by staining when staining the sample it is important to minimize the amount of time that the sample is exposed to aqueous solutions a typical staining protocol would include fixation prior to staining and dehydration afterwards a basic fix stain protocol is outlined in box 1 and can be used as a starting point immunostaining is also compatible with lmd see the featured publications page 12 pat stockton who runs a core facility for laser microdissection at the nih has optimized her work flow according to the two vital criteria best stain and shortest overall time allowed from start to nucleic acid extraction she usually stain`s frozen tissue samples with cresyl violet cva for gene expression profiling boxes a and b show the exact protocols for this staining and the whole laser dissection procedure this optimized protocol has been the basis for several successful studies and publications4 using the cellcut graphical interface to identify cells of interest cut them out using the mmi high precision and low power uv laser collecting the sample using an adhesive cap and processing the sample in the attached pcr tube is tip a very simple process that takes the least amount of time of the mmi offers a highly approved whole experiment when using the instrument allow a small buffand ready to use h e staining kit ­ rnase-free 70301 er about as wide as the cut path between the cell of interest and you simply pipet from drop bottles the laser cut so that uv energy is not absorbed into the surrounding tissue successful results can be achieved from as few as 10 cells however to improve your chances of success it is recommended to use 500 cells or more if possible especially for beginners5 as a general rule for calculation purposes you can expect to recover ~10 pg of total rna from one cell use the mmi area calculation tool to get an idea about the sections sizes you recovery and an estimate of the number of cells that you dissected this number is highly variable based on tissue type sample thickness tissue quality etc and should only be used a guide adjusting the mmi high precision and low power uv laser is easy and convinient b cva solution 0.5 g cresyl violet acetate sigma-aldrich 50 ml nuclease-free water make fresh and filter 1 75 etoh 1 minute 2 nuclease-free water 30 seconds 3 cresyl violet filtered stain 30 seconds 4 75 etoh 30 seconds 5 95 etoh 30 seconds 6 100 etoh 30 seconds x2 rna extraction purification quality control there are many kits by many manufacturers that can be used effectively with lmd samples the most popular extraction method is to use a gitc guanidinium isothiocyanate based extraction buffer and purification columns these types of kits are common in rna laboratories when selecting a kit choose one that elutes into a small volume of elution buffer or water 10 to 20 ul as you will have a small amount of rna due to the nature of the experiment for comparative qpcr experiments it is imperative to check the quantity the quality and the integrity of the rna preparation rna yields are very low after laser microdissection but the nanodrop spectrophotometric technology is an excellent choice to assess rna quantities down to 2ng per l this sensitive detection limit is not within the resolution of an agilent bioanalyzer system which is at 25 ng per l using ribogreen dye to fluorescently label rna in combination with a fluorospectrometer of type nanodrop 3300 even scales down the measurement sensitivity to 25 ng per ml or 25 pg per ul nevertheless in this work flow the appropriate agilent bioanalyzer chip ­ either the nano or the pico chip ­ give valuable information on the rna quality and integrity 4 gohlke m.j stockton s.p sieber s foley j portier j 2009 ahr mediated gene expression in the developing mouse telencephalon reproductive toxicology 28 312 328 www.molecular-machines.com mmi-bibliography 10

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»lead the way to scientific references« mmi reports by displaying the so called rin number as shown in the figure below the agilent bioanalyzer chips have a qualitative range of 50 ­ 5.000 pg per ml for the pico chip and of 5 ­ 500ng per ml for the nano chip rin numbers greater than 5 are considered good but results could be obtained from samples showing even lower rin numbers rna amplification most microarray experiments will require the use of rna amplification because the rna output from your lmd experiment will likely be in the low nanogram range and the input requirements from your microarray manufacturer will likely be in the low microgram range there are two amplification methods that people are using for lmd experiments linear amplification and whole transcript amplification wta linear amplification is considered the gold standard for rna both customer reports in this issue describe linear amplification and there are many publications that support this protocol as a reliable and reproducible method for increasing rna yield the process is new software based on doing reverse transcription from the 3 end the one down side of this is that the sample will be 3 oriented and this must be taken into account when analyzing the data wta is an alternative to linear amplification that appears to becoming popular6 this is based on using a randomized set of primers that eliminates the 3 orientation seen in linear amplification as this technology is new there are not as many publications to support this amplification however this does appear to be gaining popularity and may be the preferred amplification method in the future only time will tell when designing a genetic experiment the most critical factor is to ensure that the sample has good rna in before beginning the experiment proper sample acquisition and sample assessment are critical as the whole experiment can be compromised if these are not performed properly the instrument itself while critical actually is the smallest expense of time in the process extraction amplification labeling and analysis are reliable and proven rna methods that can be easily incorporated into your lmd experiments as long as you are working with a high quality sample and the correct precautions are taken regarding the low amount of material that will be collected then your existing staining and analysis methods can easily be incorporated into an lmd experiment 5 espina w wulfkuhle j.d calvert v.s vanmeter a zhou w coukus g geha d.h petricoin e.f and liotta l.a 2006 laser-capture microdissection nat protoc 1 586 -603 6 tomlins a.s mehra r rhodes r.d shah b.r rubin a m bruening e makarov v chinnaiyan m.a 2006 whole transcriptome amplification for gene expression profiling and development of molecular archives neoplasia vol 8 153-162 optimized protocol c protocol for laser microdissection by pat stockton wear gloves at all times and change frequently from frozen tissue 1 cut 4 membrane slides containing two sections per slide at 8 µm 2 cut 1 map h&e at 6 µm for coverslipping and diagnosis 3 hold slides on dry ice to stain in batches for lm 4 rna isolation and rt-pcr to microarray analysis by investigator briefly 1 thaw the nucleic acid extraction buffer before using 2 quick stain with fresh 1 aqueous cva filtered 3 air dry for 5 minutes under hood 4 prepare mmi laser microscope and sample tubes during the 5 minute drying 5 find target areas and microdissect all samples within 20 minutes for best rna recording the session with images saved as you progress 6 as tubes are finished pipet in the rna extraction buffer closing tubes to shake down the buffer covering your lm samples on the cap vortex the inverted tubes briefly and leave inverted until all tubes are collected or dilute the sample by pipetting up and down on the adhesive lid 7 follow the detailed protocol of the kit supplier 8 spin down gently to button the lysed rna for -80º c storage until rna isolation is performed rapid isolation is best long freezer storage is not recommended 9 check by nanodrop and picochip in the microarray lab to quantify rna start by staining and microdissecting one slide and work up your number and speed to do three to five slides in the 20 minute time period www.molecular-machines.com mmi-bibliography 11

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»lead the way to micromanipulation« f e at uredpublic at ions featured publications rna quality control results for the studies of bladder dysfunctions see maake et al sonne s b dalgaard d m nielsen e j hoei-hansen e c rajpert-de meyts e gjerdum l m leffers h 2009 optimizing staining protocols for laser microdissection of specific cell types from the testis including carcinoma in situ plos one may 2009 may vol4 5 e5536 sonne s b almstrup k dalgaard m juncker a s edsgard d ruban l harrison n .j schwager c abdollahi a huber p e brunak s gjerdrum l m moore h d andrews p w skakkebaek n e rajpert-de meyts e leffers h 2009 analysis of gene expression profiles of microdissected cell populations indicates that testicular carcinoma in situ is an arrested gonocyte cancer res 2009 jun 15;6912 5241-50 the accompanying methods article to the publication in cancer research 2009 has been published in the open access journal plos one and provides all interested scientists with a comprehensive overview on different staining and fixation methods for fresh frozen and ffpe protocols almost all commercially available kits were tested and results were summarized according to rin numbers measured with an agilent pico kit all lmd experiments were performed on an mmi cellcutplus system buckanovich r j sasaroli d o`brien-jenkins a bothbyl j conejo-garcia j r benencia f liotta l a gimotty p a and coukos g 2006 use of immuno-lcm to identify the in situ expression profile of cellular constituents of the tumor microenvironment cancer biol ther 5 635-642 a research publication leading the way to the identification of new biomarkers in the tumor environment by transcriptional expression profiling combining lmd with immune-lcm a very detailed methods part with excellent pictures and explanations of all necessary titration steps an optimized immunostaining prot ocol is introduced that takes 25 min and works with a range of commercially available antibodies duration between lmd start of rna extraction was 3 hours max without significant loss or degradation of rna in agreement with the findings by sonne et al ­ see above ­ acetone was among the best fixatives for fresh frozen material john h wlach m lehmann t maake m 2009 connexin45 expression in the human obstructed detrusor muscle world j urol 2009 27:411­418 a recent publication by dr maakes group at the university of zurich for the first time this work provides molecular evidence that in patients who suffer from bladder outlet obstruction due to benign prostate hyperplasia the expression of connexin 45 can be precisely assigned to the detrusor muscle cells whereas connexin43 expression can be assigned to the surrounding stroma both tissue compartments were microdissected and analyzed by qrtpcr separately this work also illustrates that particular tissue structures can be resolved and targeted by laser microdissection without any further staining of a tissue section as shown in figure 1 of the publication zembutsu h suzuki y sasaki a tsunoda t okazaki m yoshimoo m hasegawa t hirata k nakamura y 2008 epub ahead predicting response to docetaxel neoadjuvant chemotherapy for advanced breast cancers through genome-wide expression profiling international journal of oncology 2009 34 361-370 one of the first studies that integrated laser microdissection into a clinical study in order to predict breast cancer patient outcome under therapy ­ here with docetaxel this group analyzed gene expression profiles of biopsy materials from 29 advanced breast cancers using a cdna microarray consisting of 36,864 genes or ests after enrichment of cancer cell population with the mmi cellcutplus laser microdissection system analyzing eight pr partial response patients and twelve patients with sd stable disease or pd progressive disease response the group identified dozens of genes that were expressed differently between the `responder pr and `nonresponder sd or pd groups a selection of nine `predictive genes showing the most significant differences together with a numerical prediction scoring system that clearly separated the responder group from the non-responder group was developed www.molecular-machines.com mmi-bibliography 12 grafical and tabular display of an agilent bioanalyzer run sample 6 contains 1093 pg/ul rna and has a rin number of 6.1 courtesy of dr maake university of zurich expression of smoothelin variants in the human detrusor

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»lead the way to scientific references« references agarwal n lippmann e s shusta e v 2010 ashida s furihata m katagiri t tamura k anazawa y yoshioka h miki t fujioka t shuin t nakamura y and nakagawa h 2006 ashida s nakagawa h katagiri t furihata m iiizumi m anazawa y tsunoda t takata r kasahara k miki t fujioka t shuin t and nakamura y 2004 bohm m wieland i schutze k and rubben h 1997 buckanovich r j sasaroli d o`brien-jenkins a botbyl j conejo-garcia j r benencia f liotta l a gimotty p a and coukos g 2006 buckanovich r j sasaroli d o`brien-jenkins a botbyl j hammond r katsaros d sandaltzopoulos r liotta l a gimotty p a and coukos g 2007 cowherd s m espina v a petricoin e f iii and liotta l a 2004 cunnea p mcmahon j o`connell e mashayekhi k fitzgerald u mcquaid s 2009 dahl e kristiansen g gottlob k klaman i ebner e hinzmann b hermann k pilarsky c durst m klinkhammerschalke m blaszyk h knuechel r hartmann a rosenthal a and wild p.j 2006 erickson s h albert s p gillespie w j canales-rodriguez j linehan m w pinto a p chuaqui f r emmert-buck r m 2009 espina v dettloff k a cowherd s petricoin e f iii and liotta l a 2004 espina v heiby m pierobon m and liotta l a 2007 espina v wulfkuhle j d calvert v s vanmeter a zhou w coukos g geho d h petricoin e f iii and liotta l a 2006 fevr t robine s louvard d and huelsken j 2007 gao n lu m echeverri f laita b kalabat d et al 2009 gao n lu m echeverri f laita b kalabar d wlilliams e m hevezi p zlotnik a moyer d b 2009 gjerdrum l m abrahamsen h n villegas b sorensen b s schmidt h and hamilton-dutoit s j 2004 gjerdrum l m and hamilton-dutoit s 2005a gjerdrum l.m and hamilton-dutoit s 2005b gjerdrum l m lielpetere i rasmussen l m bendix k and hamilton-dutoit s 2001 gohlke m j stockton s p sieber s foley j portier j 2009 harrel c j dye w w harvell e m d sartorius a c horwitz b k 2008 hatakeyama h kondo t fujii k nakanishi y kato h fukuda s and hirohashi s 2006 identification and expression profiling of blood-brain barrier membrane proteins journal of neurochemistry 2010 112 625-635 expression of novel molecules mical2-pv mical2 prostate cancer variants increases with high gleason score and prostate cancer progression clin cancer res 12 2767-2773 molecular features of the transition from prostatic intraepithelial neoplasia pin to prostate cancer genome-wide gene-expression profiles of prostate cancers and pins cancer res 64 5963-5972 microbeam moment non-contact laser microdissection of membrane-mounted native tissue am j pathol 151 63-67 use of immuno-lcm to identify the in situ expression profile of cellular constituents of the tumor microenvironment cancer biol ther 5 635-642 tumor vascular proteins as biomarkers in ovarian cancer j clin oncol 25 852-861 proteomic analysis of human breast cancer tissue with laser-capture microdissection and reversephase protein microarrays clin breast cancer 5 385-392 gene expression analysis of the microvascular compartment in multiple sclerosis using laser microdissected blood vessels acta neuropathol 2010 119 601-615 molecular profiling of laser-microdissected matched tumor and normal breast tissue identifies karyopherin alpha2 as a potential novel prognostic marker in breast cancer clin cancer res 12 3950-3960 quantitative rt-pct gene expression analysis of laser microdissected tissue samples nat protoc.2009 4 902-922 use of proteomic analysis to monitor responses to biological therapies expert opin biol ther 4 83-93 laser capture microdissection technology laser-capture microdissection expert rev mol diagn 7 647-657 nat protoc 1 586-603 wnt/beta-catenin is essential for intestinal homeostasis and maintenance of intestinal stem cells mol cell biol 27 7551-7559 voltage-gated sodium channels in taste bud cells voltage-gated sodium channels in taste bud cells bmc neurosci 2010 10 20 bmc neuroscience 10:20 the influence of immunohistochemistry on mrna recovery from microdissected frozen and formalin-fixed paraffin-embedded sections diagn mol pathol 13 224-233 laser-assisted microdissection of membrane-mounted sections following immunohistochemistry and in situ hybridization methods mol biol 293 139-149 laser-assisted microdissection of membrane-mounted tissue sections methods mol biol 293 127-138 laser-assisted microdissection of membrane-mounted paraffin sections for polymerase chain reaction analysis identification of cell populations using immunohistochemistry and in situ hybridization j mol diagn 3 105-110 ahr-mediated gene expression in the developing mouse telencephalon reproductive toxicology 28 312 328 contaminating cells alter gene signatures in whole organ versus laser capture microdissected tumors a comparison of experimental breast cancers and their lymph node metastases clin exp metastasis 2008 25 1 81-8 protein clusters associated with carcinogenesis histological differentiation and nodal metastasis in esophageal cancer proteomics 6 6300-6316 www.molecular-machines.com mmi-bibliography 13

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»lead the way to scientific references« references hlubek f schuster c budczies j kirchner t 2010 hevezi p moyer b d lu m gao n white e et al 2009 hlubek f brabletz t budczies j pfeiffer s jung a kirchner t 2007 john h walch m lehmann t maake c 2009 jorgensen a nielsen j e morthorst j e bjerregaard p leffers h 2009 kasper g vogel a klaman i grone j petersen i weber b castanos-velez e staub e and mennerich d 2005a kasper g weiser a a rump a sparbier k dahl e hartmann a wild p schwidetzky u castanos-velez e and lehmann k 2005b kondo t and hirohashi s 2006 robust microrna expression profiling of specific cells in complex archival tissue stained by immunohistochemistry abstract at aacr 2010 and paper in submitted genome-wide analysis of gene expression in primate taste buds reveals links to diverse processes plos one 2009 4 e6395 heterogeneous expression of wnt/ß-catenin target genes within colorectal cancer int j cancer 121 1941-1948 connexin 45 expression in the human obstructed detrusor 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»lead the way to scientific references« references scarpino s di n a rapazzotti-onelli m pilozzi e and ruco l 2004b scarpino s di n a stoppacciaro a antonelli m pilozzi e chiarle r palestro g marino m facciolo f rendina e a webster k e kinkel s a scott h s and ruco l 2007b scarpino s di n a taraboletti g cancrini a and ruco l p 2005 schipper h papp t johnen g pemsel h bastrop r muller k m wiethege t jaworska m krismann m schiffmann d and rahman q 2003 sonne s b dalgaard d m nielsen e j hoei-hansen e c rajpert-de meyts e gjerdum l m leffers h 2009 sonne s b almstrup k dalgaard m juncker a.s edsgard ,d ruban l harrison n.j schwager ,c abdollahi a huber p e brunak s gjerdrum l m moore h d andrews p w skakkebaek n e rajpert-de meyts e leffers h 2009 tanney a kennedy r d 2010 takata r katagiri t kanehira m tsunoda t shuin t miki t namiki m kohri k matsushita y fujioka t and nakamura y 2005 tamura k furihata m tsunoda t ashida s takata r obara w yoshioka h daigo y nasu y kumon h konaka h namiki m tozawa k kohri k tanji n yokoyama m shimazui t akaza h mizutani y miki t fujioka t shuin t nakamura y and nakagawa h 2007 tomlins s a mehra r rhodes d r cao x wang l dhanasekaran s m kalyana-sundaram s wei j t rubin m va pienta k j shah r.vb and chinnaiyan a m 2007 tomlins s a mehra r rhodes d r shah r b rubin m a bruening e makarov v and chinnaiyan a m 2006 tveito s maelandsmo g m hoifodt h k rasmussen h and fodstad o 2007 wang l and zhu h 2006 wu m han l shi y xu g wei j you l chen y zhu t li s meng l lu y zhou j wang s ma d 2009 yamabuki t daigo y kato t hayama s tsunoda t miyamoto m ito t fujita m hosokawa m kondo s and nakamura y 2006 zembutsu h suzuki y sasaki a tsunoda t okazaki m yoshimoo m hasegawa t hirata k nakamura y 2008 epub ahead zhang l yang n conejo-garcia j r katsaros d mohamed-hadley a fracchioli s schlienger k toll a levine b rubin s c and coukos g 2003a zhang l yang n park j.w katsaros d fracchioli s cao g o`brien-jenkins a randall t c rubin s c and coukos g 2003b papillary carcinoma of the thyroid methylation is not involved in the regulation of met expression br j cancer 91 703-706 expression of autoimmune regulator gene aire and t regulatory cells in human thymomas clin exp immunol 149 504-512 hepatocyte growth factor hgf downregulates thrombospondin 1 tsp-1 expression in thyroid papillary carcinoma cells j pathol 205 50-56 mutational analysis of the nf2 tumour suppressor gene in three subtypes of primary human malignant mesotheliomas int j oncol 22 1009-1017 optimizing staining protocols for laser microdissection of specific cell types from the testis including carcinoma in situ plos one may 2009 may vol4 5e5536 analysis of gene expression profiles of microdissected cell populations indicates that testicular carcinoma in situ is an arrested gonocyte cancer res 2009 jun 15;6912 5241-50 developing mrna based biomarkers from formalin-fixed paraffin-embedded tissue personalized medicine 2010 72 205-211 predicting response to methotrexate vinblastine doxorubicin and cisplatin neoadjuvant chemotherapy for bladder cancers through genome-wide gene expression profiling clin cancer res 11 2625-2636 molecular features of hormone-refractory prostate cancer cells by genome-wide gene expression profiles cancer res 67 5117-5125 integrative molecular concept modeling of prostate cancer progression nat genet 39 41-51 whole transcriptome amplification for gene expression profiling and development of molecular archives neoplasia 8 153-162 specific isolation of disseminated cancer cells a new method permitting sensitive detection of target molecules of diagnostic and therapeutic value clin exp metastasis 24 317-327 clonal analysis of palmar fibromatosis a study whether palmar fibromatosis is a real tumor j transl med 4 21 development and characterization of a novel method for the analysis of gene expression patterns in lymphatic endothelial cells derived from primary breast tissues j cancer res clin oncol 2009 doi:10.1007/s00432-009-0727-9 published online november 2009 genome-wide gene expression profile analysis of esophageal squamous cell carcinomas int j oncol 28 1375-1384 predicting response to docetaxel neoadjuvant chemotherapy for advanced breast cancers through genome-wide expression profiling international journal of oncology 2009 34 361-370 expression of endocrine gland-derived vascular endothelial growth factor in ovarian carcinoma clin cancer res 9 264-272 tumor-derived vascular endothelial growth factor up-regulates angiopoietin-2 in host endothelium and destabilizes host vasculature supporting angiogenesis in ovarian cancer cancer res 63 3403-3412 to be continued www.molecular-machines.com mmi-bibliography 15

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