mRNA Expression Profiling of Laser Microbeam Microdissected Cells from Slender Embryonic Structures
2002; Elsevier BV; Volume: 160; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)64903-6
ISSN1525-2191
AutoresStefan Scheidl, S K Nilsson, Mattias Kalén, Mats Hellström, Minoru Takemoto, Joakim Håkansson, Per Lindahl,
Tópico(s)Gene expression and cancer classification
ResumoMicroarray hybridization has rapidly evolved as an important tool for genomic studies and studies of gene regulation at the transcriptome level. Expression profiles from homogenous samples such as yeast and mammalian cell cultures are currently extending our understanding of biology, whereas analyses of multicellular organisms are more difficult because of tissue complexity. The combination of laser microdissection, RNA amplification, and microarray hybridization has the potential to provide expression profiles from selected populations of cells in vivo. In this article, we present and evaluate an experimental procedure for global gene expression analysis of slender embryonic structures using laser microbeam microdissection and laser pressure catapulting. As a proof of principle, expression profiles from 1000 cells in the mouse embryonic (E9.5) dorsal aorta were generated and compared with profiles for captured mesenchymal cells located one cell diameter further away from the aortic lumen. A number of genes were overexpressed in the aorta, including 11 previously known markers for blood vessels. Among the blood vessel markers were endoglin, tie-2, PDGFB, and integrin-β1, that are important regulators of blood vessel formation. This demonstrates that microarray analysis of laser microbeam micro-dissected cells is sufficiently sensitive for identifying genes with regulative functions. Microarray hybridization has rapidly evolved as an important tool for genomic studies and studies of gene regulation at the transcriptome level. Expression profiles from homogenous samples such as yeast and mammalian cell cultures are currently extending our understanding of biology, whereas analyses of multicellular organisms are more difficult because of tissue complexity. The combination of laser microdissection, RNA amplification, and microarray hybridization has the potential to provide expression profiles from selected populations of cells in vivo. In this article, we present and evaluate an experimental procedure for global gene expression analysis of slender embryonic structures using laser microbeam microdissection and laser pressure catapulting. As a proof of principle, expression profiles from 1000 cells in the mouse embryonic (E9.5) dorsal aorta were generated and compared with profiles for captured mesenchymal cells located one cell diameter further away from the aortic lumen. A number of genes were overexpressed in the aorta, including 11 previously known markers for blood vessels. Among the blood vessel markers were endoglin, tie-2, PDGFB, and integrin-β1, that are important regulators of blood vessel formation. This demonstrates that microarray analysis of laser microbeam micro-dissected cells is sufficiently sensitive for identifying genes with regulative functions. The recently published drafts of the human genome sequence roughly define the complement of mammalian genes.1Venter JC Adams MD Myers EW Li PW Mural RJ Sutton GG Smith HO Yandell M Evans CA Holt RA Gocayne JD Amanatides P Ballew RM Huson DH Wortman JR Zhang Q Kodira CD Zheng XH Chen L Skupski M Subramanian G Thomas PD Zhang J Gabor Miklos GL Nelson C Broder S Clark AG Nadeau J McKusick VA Zinder N Levine AJ Roberts RJ Simon M Slayman C Hunkapiller M Bolanos R Delcher A Dew I Fasulo D Flanigan M Florea L Halpern A Hannenhalli S Kravitz S Levy S Mobarry C Reinert K Remington K Abu-Threideh J Beasley E Biddick K Bonazzi V Brandon R Cargill M Chandramouliswaran I Charlab R Chaturvedi K Deng Z Di Francesco V Dunn P Eilbeck K Evangelista C Gabrielian AE Gan W Ge W Gong F Gu Z Guan P Heiman TJ Higgins ME Ji RR Ke Z Ketchum KA Lai Z Lei Y Li Z Li J Liang Y Lin X Lu F Merkulov GV Milshina N Moore HM Naik AK Narayan VA Neelam B Nusskern D Rusch DB Salzberg S Shao W Shue B Sun J Wang Z Wang A Wang X Wang J Wei M Wides R The sequence of the human genome.Science. 2001; 291: 1304-1351Crossref PubMed Scopus (10470) Google Scholar, 2Lander ES Linton LM Birren B Nusbaum C Zody MC Baldwin J Devon K Dewar K Doyle M FitzHugh W Funke R Gage D Harris K Heaford A Howland J Kann L Lehoczky J LeVine R McEwan P McKernan K Meldrim J Mesirov JP Miranda C Morris W Naylor J Raymond C Rosetti M Santos R Sheridan A Sougnez C Stange-Thomann N Stojanovic N Subramanian A Wyman D Rogers J Sulston J Ainscough R Beck S Bentley D Burton J Clee C Carter N Coulson A Deadman R Deloukas P Dunham A Dunham I Durbin R French L Grafham D Gregory S Hubbard T Humphray S Hunt A Jones M Lloyd C McMurray A Matthews L Mercer S Milne S Mullikin JC Mungall A Plumb R Ross M Shownkeen R Sims S Waterston RH Wilson RK Hillier LW McPherson JD Marra MA Mardis ER Fulton LA Chinwalla AT Pepin KH Gish WR Chissoe SL Wendl MC Delehaunty KD Miner TL Delehaunty A Kramer JB Cook LL Fulton RS Johnson DL Minx PJ Clifton SW Hawkins T Branscomb E Predki P Richardson P Wenning S Slezak T Doggett N Cheng JF Olsen A Lucas S Elkin C Uberbacher E Frazier M Gibbs RA Muzny DM Scherer SE Bouck JB Sodergren EJ Worley KC Rives CM Gorrell JH Metzker ML Naylor SL Kucherlapati RS Nelson DL Weinstock GM Sakaki Y Fujiyama A Hattori M Yada T Toyoda A Itoh T Kawagoe C Watanabe H Totoki Y Taylor T Weissenbach J Heilig R Saurin W Artiguenave F Brottier P Bruls T Pelletier E Robert C Wincker P Smith DR Doucette-Stamm L Rubenfield M Weinstock K Lee HM Dubois J Rosenthal A Platzer M Nyakatura G Taudien S Rump A Yang H Yu J Wang J Huang G Gu J Hood L Rowen L Madan A Qin S Davis RW Federspiel NA Abola AP Proctor MJ Myers RM Schmutz J Dickson M Grimwood J Cox DR Olson MV Kaul R Shimizu N Kawasaki K Minoshima S Evans GA Athanasiou M Schultz R Roe BA Chen F Pan H Ramser J Lehrach H Reinhardt R McCombie WR de la Bastide M Dedhia N Blocker H Hornischer K Nordsiek G Agarwala R Aravind L Bailey JA Bateman A Batzoglou S Birney E Bork P Brown DG Burge CB Cerutti L Chen HC Church D Clamp M Copley RR Doerks T Eddy SR Eichler EE Furey TS Galagan J Gilbert JG Harmon C Hayashizaki Y Haussler D Hermjakob H Hokamp K Jang W Johnson LS Jones TA Kasif S Kaspryzk A Kennedy S Kent WJ Kitts P Koonin EV Korf I Kulp D Lancet D Lowe TM McLysaght A Mikkelsen T Moran JV Mulder N Pollara VJ Ponting CP Schuler G Schultz J Slater G Smit AF Stupka E Szustakowki J Thierry-Mieg D Wagner L Wallis J Wheeler R Williams A Wolf YI Wolfe KH Yang SP Yeh RF Collins F Guyer MS Peterson J Felsenfeld A Wetterstrand KA Patrinos A Morgan MJ Initial sequencing and analysis of the human genome.Nature. 2001; 409: 860-921Crossref PubMed Scopus (17501) Google Scholar Consequently, analyses of cell behavior in the context of gene expression patterns will be increasingly valuable. Global expression studies are most straightforward in systems with large and accessible populations of equivalent cells such as yeast or mammalian cell cultures. Valuable results have also been generated with heterogeneous samples such as cancer tumors and whole Drosophila embryos,3Alizadeh AA Eisen MB Davis RE Ma C Lossos IS Rosenwald A Boldrick JC Sabet H Tran T Yu X Powell JI Yang L Marti GE Moore T Hudson Jr, J Lu L Lewis DB Tibshirani R Sherlock G Chan WC Greiner TC Weisenburger DD Armitage JO Warnke R Levy R Wilson W Grever MR Byrd JC Botstein D Brown PO Staudt LM Distinct types of diffuse large B-cell lymphoma identified by gene expression profiling.Nature. 2000; 403: 503-511Crossref PubMed Scopus (7919) Google Scholar, 4White KP Rifkin SA Hurban P Hogness DS Microarray analysis of Drosophila development during metamorphosis.Science. 1999; 286: 2179-2184Crossref PubMed Scopus (376) Google Scholar but the sample complexity sets a limit as to what kind of questions might be addressed. The expression profile of certain cells or cell types cannot be resolved, and differences between samples might reflect different cell compositions rather than different transcript abundance. Also, the sensitivity of cell-specific transcripts is reduced as these mRNA are diluted with transcripts from other cell types. Methods to isolate homogenous samples in vivo must be adopted and refined to fully exploit the potential of expression profiles in mammalian studies.In this article we present a procedure for microarray hybridization of RNA extracted from cells isolated with laser microbeam microdissection (LMM) and laser pressure catapulting (LPC).5Schutze K Lahr G Identification of expressed genes by laser-mediated manipulation of single cells.Nature Biotechnol. 1998; 16: 737-742Crossref Scopus (371) Google Scholar This procedure offers several important advantages compared to previously used microdissection methods:6Luo L Salunga RC Guo H Bittner A Joy KC Galindo JE Xiao H Rogers KE Wan JS Jackson MR Erlander MG Gene expression profiles of laser-captured adjacent neuronal subtypes.Nat Med. 1999; 5: 117-122Crossref PubMed Scopus (643) Google Scholar, 7Kitahara O Furukawa Y Tanaka T Kihara C Ono K Yanagawa R Nita ME Takagi T Nakamura Y Tsunoda T Alterations of gene expression during colorectal carcinogenesis revealed by cDNA microarrays after laser-capture microdissection of tumor tissues and normal epithelia.Cancer Res. 2001; 61: 3544-3549PubMed Google Scholar, 8Ono K Tanaka T Tsunoda T Kitahara O Kihara C Okamoto A Ochiai K Takagi T Nakamura Y Identification by cDNA microarray of genes involved in ovarian carcinogenesis.Cancer Res. 2000; 60: 5007-5011PubMed Google Scholar the sample is not in contact with any part of the equipment or the collector device during the isolation process thus dramatically reducing the risk of contamination. LMM also allows cells of any shape and size (down to pieces of chromosomes) to be cut out and catapulted. Laser microdissection offers precise control as the cells are picked individually from histological sections. No cell-specific markers are needed for the cell isolation, even populations that are recognized by morphology alone can be isolated purely. Contrary to fluorescence-activated cell sorting or magnetic bead sorting, the tissues are not exposed to collagenase digestion before cell isolation, but are fixed or frozen in their native environment conserving the RNA profile in a true in vivo state. We are primarily interested in cell fate decisions and differentiation that typically occurs in clusters of cells located in slender embryonic structures.9Wolpert L Principles of Development. Oxford University Press, New York1998Google Scholar As these structures are not preserved without fixation the procedure was optimized for fixed material, which in turn makes it applicable to most cell populations. As a proof of principle, expression profiles from cells in the mouse embryonic dorsal aorta at the onset of vascular smooth muscle cell (VSMC) induction was compared to the expression profile from mesenchymal cells located one cell diameter further away from the aorta lumen. Genes encoding endothelial markers, smooth muscle cell markers, and basement membrane proteins, were consistently overexpressed in the aorta cells, confirming the accuracy of the profiles. No such markers were overexpressed in the mesenchymal cells.Materials and MethodsMiceC57BL/6 mice were housed at the Department of Experimental Biomedicine at Göteborg University according to Swedish animal research regulations. All experiments have been approved by the Swedish Research Animal Ethical Committee (Drnr: 213-2000). The morning of vaginal plug detection was counted as E0.5.FixationThe following fixatives were tested: zinc-fix (5 g ZnCl2, 6 g ZnAc2 × 2H2O, 0.1 g CaAc2, in 1 L of 0.1 mol/L Tris, pH 7.4), methanol, 70% ethanol, acetone, 4% paraformaldehyde, Formoys (60 ml EtOH, 10 ml HAc, 30 ml of 40% formaldehyde), Carnoys (50 ml EtOH, 25 ml HAc), and methacarn (60 ml EtOH, 30 ml chloroform, 10 ml HAc). Animals were dissected in ice-cold phosphate-buffered saline. Tissues were immersed in respective fixative and left overnight at 4°C.RNA Recovery and Quality MeasurementsTotal RNA was extracted from P14 mouse kidneys and hearts with the Qiagen RNeasy mini kit (VWR International AB, Stockholm, Sweden). RNA content was quantified with UV-spectrophotometric analysis (A260), and recovery rates are presented as percentage of RNA content in directly homogenized tissue. RNA integrity was analyzed with electrophoresis using the NorthernMax kit (Ambion Ltd, Cambridgeshire, UK). Five μg of total RNA was loaded on 1% agarose gels. The RNA quality was evaluated by incorporation of 32P-labeled CTP in the first and second strand cDNA-synthesis reaction. cDNA was generated with the replacement method according to standard protocols using a polydT primer. 32P-labeled CTP was added to a final concentration of 1 μCi/μl of 10 mmol/L dNTP mix. The32P-labeled cDNA was size-fractionated on a 0.8% agarose gel, transferred to nylon filter, and analyzed with a phosphoimager according to standard procedures.Small amounts (<1 μg) of total RNA were extracted with the Micro RNA Isolation Kit (Zymo Research, Orange, CA). RNA quantities were measured with Ribo-Green RNA Quantitation Kit (Molecular Probes Europe BV, Leiden, The Netherlands) in a fluorometer (TD-360; Turner Designs Inc, Sunnyvale, CA). All procedures were performed according to the manufacturers' instructions.LMM and LPCE9.5 mouse embryos (C57BL/6) were zinc-fixed (4°C) overnight, and then incubated 4 hours in zinc-fix with 30% sucrose. Embryos were mounted in Tissue-Tek OCT compound (Sakura Finetek, Torrence, CA) and stored at −80°C until sectioning. Frozen sections (10 μm) were mounted on plus-charged slides (SuperFrost plus; Menzel-Gläser, Braunschweig, Germany), and left to dry for 30 minutes in room temperature before storage at −80°C in boxes with silica gel. Every sixth section was mounted on a reference glass that was stained for α-smooth muscle actin (α-SMA). For laser capturing, the slides were put into zinc-fix on ice for 5 minutes to dissolve the OCT that otherwise interferes with LMM. Next, the slides were dehydrated for 30 seconds in 70%, 95%, and 99.5% ice-cold ethanol, respectively, incubated 1 minute in xylene, and dried at room temperature. Five sections were mounted on each slide and were captured in one session using the PALM Robot-MicroBeam system (P.A.L.M. Mikrolaser Technology AG, Bernried, Germany). The LPC-collected cells (∼200) were solved in 40 μl of lysis buffer (Micro RNA isolation kit; Zymo) and stored at −80°C. The process was repeated until 1000 cells were collected.RNA Extraction and T7 RNA AmplificationLMM-isolated cells in lysis buffer were thawed and centrifuged briefly before the RNA was extracted using the Micro RNA isolation kit (Zymo) according to the manufacturer's protocol. T7 RNA (aRNA) amplification was performed in three cycles mainly according to the protocol given in Wang and colleagues10Wang E Miller LD Ohnmacht GA Liu ET Marincola FM High-fidelity mRNA amplification for gene profiling.Nature Biotechnol. 2000; 18: 457-459Crossref Scopus (578) Google Scholar but with the following modifications: 0.5 μl (5 μg/μl) of linear acrylamide (Ambion Ltd.) was added in the first step of oligo-dT(15)-T7 primer annealing. After second-strand synthesis, double-stranded cDNA was phenol-chloroform-isoamylalcohol extracted once and washed three times with RNase-free water (Ambion Ltd.) on Microcon 100 columns (Millipore AB, Sundbyberg, Sweden). The final volume was adjusted to 16 μl. After in vitro transcription with the T7 Megascript Kit (Ambion Ltd.) for 4 hours at 37°C, the reaction mixture was mixed with 460 μl of lysis buffer (GeneElute kit; Sigma-Aldrich Chemie GmbH, Munich, Germany) and the aRNA was purified according to the RNA isolation protocol provided by the manufacturer. aRNA was eluted from the column with 50 μl of water and vacuum-dried in the presence of 60 U of RNasin (Promega UK, Southampton, UK) to a volume of 5 μl. Subsequent rounds of amplification were performed as described elsewhere.10Wang E Miller LD Ohnmacht GA Liu ET Marincola FM High-fidelity mRNA amplification for gene profiling.Nature Biotechnol. 2000; 18: 457-459Crossref Scopus (578) Google Scholar However, cDNA purification, in vitro transcription, and aRNA purification were performed as for the first round. A detailed protocol can be downloaded from http://cbz.medkem.gu.se/lindahl/protocols.Target Labeling and Microarray HybridizationFive μg of aRNA or 100 μg of total RNA was primed with 5 μg of random hexamer (Promega UK) or 2 μg of oligo-dT primer, respectively, and labeled in a reverse transcription reaction with Cy3-dUTP or Cy5-dUTP (Amersham Pharmacia Biotech AB, Uppsala, Sweden) in a volume of 30 μl, according to standard protocols (http://cmgm.stanford.edu/pbrown). The differently labeled targets were combined, mixed with 1 μl of 10 μg/μl yeast tRNA, 1 μl of 10 μg/μl polyA RNA, vacuum-dried, and resuspended in 20 μl of DIGeasy hybridization buffer (Roche Diagnostics GmbH, Mannheim, Germany). The hybridization mix was placed at 100°C for 2 minutes and then at 37°C for 30 minutes before being added to the chip. Before hybridization, the glasses were rehydrated by placing them array-side down for 15 minutes over 1× standard saline citrate (SSC), fast-dried by placing them array-side up for 10 seconds on a 100°C heat block, and then baked at 80°C for 4 hours and UV cross-linked (300 mJ).Hybridization was performed in a 40°C water bath for 12 to 18 hours under lifter coverslip (Histolab, Göteborg, Sweden) in ArrayIT hybridization cassettes (TeleChem International Inc, Sunnyvale, CA). After hybridization, the slides were washed in 2× SSC, 0.1% sodium dodecyl sulfate for 5 minutes at room temperature, 1× SSC for 5 minutes, and finally in 0.1× SSC for 5 minutes.Array Scanning and Data PresentationThe slides were scanned (ScanArray 3000; Packard Bioscience, Meriden, CT) at laser intensity and photomultiplier tube voltage settings giving the best dynamic range for each chip in respective channel. Image segmentation and spot quantification was performed with the ImaGene software (Biodiscovery, Marina Del Rey, CA). After median local background subtraction, the log2-transformed ratios (Cy3intensity/Cy5intensity) were plotted versus the mean log2 intensities 0.5*(log2Cy3intensity + log2Cy5intensity). The ratios were normalized for signal intensity variation in a non-linear intensity-dependent way using the loess function in the S-Plus software (MathSoft Inc, Surrey, UK) as described elsewhere (Dudoit S, Yang YH, Callow MJ, Speed TP, technical report no. 578, August 2000, Department of Biochemistry, Stanford University School of Medicine). A ratio versus intensity plot amounts to a 45° rotation of a log2Cy3intensityversus log2Cy5intensity plot, followed by scaling of the coordinates. This representation allows for intensity-dependent nonlinear normalization, but it also directly visualizes the actual expression ratios throughout the entire 16-bit range of the scanned microarray images.Statistic AnalysisMean log2-transformed ratios were calculated from repeated independent experiments. The genes were individually evaluated for overexpression by t-tests, which is applicable because the log-transformed ratios are approximately normally distributed.Evaluation of T7 AmplificationExpression profiles were generated from nonamplified and amplified samples in the following way: the amplified heart and kidney RNA was compared in four hybridization experiments using the independently amplified samples (hybridizations A1 to A4). Similarly, the nonamplified heart and kidney total RNA was compared in four independent hybridizations (hybridizations T1 to T4). Mean log2-transformed expression ratios were calculated for both categories (see Figure 3, a, e and f).For evaluation of the amplification effect on the relative abundance of transcripts within a sample (preservation of profiles), log2-transformed signal intensities of the heart channels from the nonamplified hybridizations (T1H to T4H) were compared to the heart signals from the amplified hybridizations (A1H to A4H) (see Figure 3b).For evaluation of the amplification effect on expression ratios between two samples (preservation of ratios), a ratio versus ratio plot was generated and the correlation coefficent was calculated from the mean log2-transformed expression ratios T1-4 and A1-4 (Figure 3g).To assess intensity-dependent correlation of the ratios, all individual arrays were compared, and ratio versus ratio plots for each pair were generated. Six pairs described reproducibility of nonamplified ratios (T to T comparisons). Another six pairs described reproducibility of amplified ratios (A to A comparisons). Finally, 16 plots described the correlation between nonamplified and amplified ratios (T to A comparisons). Correlations between expression ratios in the A versus A, T versus T, and T versus A pairs were calculated in an intensity-dependent way, as follows: Each ratio versus ratio plot was divided into 10 groups based on order of abundance. The 10% most abundant clones were assigned to group 1, next 10% to group 2, and so forth. The T to A plots were grouped according to order of abundance in the amplified experiments. For each group, the Pearson correlation coefficient was calculated and plotted against the log2 intensity distribution. The curves shown in Figure 3h are the loess regression lines (S-Plus software; MathSoft Inc, Surrey, UK) through the correlation coefficient plots for each category (T to T, A to A, and T to A).Microarray Chip DesignThe chips were printed with 1350 random chosen mouse sequence-verified expressed sequence tags from the I.M.A.G.E. consortium (purchased from Invitrogen Ltd, Renfrewshire, Scotland), 2400 nonsequenced clones from a normalized E16.5 head library (generously provided by Dr. Oliver Renner, Max-Planck Institute for Physiological and Clinical Research, Bad Nauheim, Germany), 5 yeast genes, and 100 selected control genes. The clones were polymerase chain reaction (PCR) amplified, purified, and resuspended in 0.2% sarkosyl, 3× SSC and printed with the GMS 417 spotter (Affimetrix, Santa Clara, CA) onto γ-amino propyl silane-coated CMT-Gap slides (Corning International, London, UK).Quantitative PCRTaqMan PCR primers and labeled probes (5′ 6-FAM, 3′ 6-TAMRA) were designed for 11 genes using the Primer Express software and purchased from Applied Biosystems (Applera Sweden, Stockholm, Sweden) (sequences can be found at http://cbz.medkem.gu.se/lindahl/taqman). The mRNA sequences were obtained from the Celera database.After laser microdissection and three rounds of RNA amplification, 60 ng of aorta and mesenchyme aRNA was used for first-strand cDNA synthesis. The aRNA was mixed with 4 μl of first strand buffer, 4 μl (500 μg/ml) of random hexamers (Promega UK), 2 μl of dithiothreitol, 1 μl of RNasin (Promega UK), 2 μl of ultrapure dNTPs mix (Clontech, Palo Alto, CA), and water to a final volume of 18 μl. The mixture was incubated at 65°C for 10 minutes followed by 42°C for 1 minute. Two μl of Superscript II (Invitrogen Ltd) was added and incubation at 42°C was continued for 1 hour.The 50-cycle TaqMan PCR assay was performed in 20-μl reactions in a 384-well microtiter plate under conditions recommended by the manufacturer using 10 μl TaqMan 2× PCR Master Mix (Applera Sweden), 6 pmol of each primer, 2 pmol of probe, and 1.5 ng of cDNA (1/40 of the cDNA reaction mix) using the AB1 PRISM 9700HT realtime PCR cycler (Applied Biosystems, Foster City, CA). Each assay was repeated three times and the mean CT-values were used for further calculations. For each amplicon a standard curve was determined using eight serial dilutions in triplicate of a mixed cDNA template obtained from heart, kidney, and brain total RNA. The relative number of target copies in each sample was interpolated from its detection threshold (CT) value using the standard curve. Expression ratios between aorta and mesenchyme target copies were calculated and changes <20-fold were plotted against the fold changes measured on the microarray. The TaqMan measured ratios were normalized with respect to the offset of the linear fit through this plot.Results and DiscussionEvaluation of FixativesThe main challenge for obtaining expression profiles from laser-microdissected cells is the limited amount of RNA that can be extracted, and it is therefore important to eliminate RNA losses at any stage. It is equally important to maintain the integrity of the transcripts because reverse transcription is a prerequisite for amplification and target labeling. Several fixatives were compared to evaluate total RNA recovery, RNA quality and the RNA performance as a template for cDNA synthesis (Figure 1; a to c). Generally, compounds with good recovery rates also preserved the integrity of the RNA and generated long cDNA products. Precipitating agents such as methanol, ethanol, and acetone efficiently recovered RNA with preserved integrity, which is in line with previous reports.11Goldsworthy SM Stockton PS Trempus CS Foley JF Maronpot RR Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue.Mol Carcinog. 1999; 25: 86-91Crossref PubMed Scopus (291) Google Scholar Cross-linking agents such as 4% paraformaldehyde and Formoys were not as favorable and failed to produce but minor amounts of RNA, also in agreement with the literature.11Goldsworthy SM Stockton PS Trempus CS Foley JF Maronpot RR Effects of fixation on RNA extraction and amplification from laser capture microdissected tissue.Mol Carcinog. 1999; 25: 86-91Crossref PubMed Scopus (291) Google Scholar Surprisingly, a zinc-based fixative performed best in our test.12Johansson M Jansson T Powell TL Na(+)-K(+)-ATPase is distributed to microvillous and basal membrane of the syncytiotrophoblast in human placenta.Am J Physiol. 2000; 279: R287-R294Google Scholar RNA was recovered almost as efficiently as after direct homogenization, and the synthesized cDNA was of comparable length. The protection against endogenous RNases was sufficient for RNA extraction from kidneys and whole embryos, but not from pancreas, suggesting that zinc-fix has a moderate protective ability (data not shown). Contrary to the literature,13Shibutani M Uneyama C Miyazaki K Toyoda K Hirose M Methacarn fixation: a novel tool for analysis of gene expressions in paraffin-embedded tissue specimens.Lab Invest. 2000; 80: 199-208Crossref PubMed Scopus (109) Google Scholar methacarn and Carnoys repeatedly produced low amounts of RNA in our hands (Figure 1; a to c).Figure 1Several fixatives were evaluated for RNA recovery (a), RNA integrity (b), and cDNA synthesis (c). a: Kidneys from P14 mice were placed in fixative at 4°C overnight. RNA recovery is presented as percent recovery of directly homogenized tissue (error bars, 1 std). b: Five μg of the extracted total RNA was loaded on a denatured agarose gel for integrity evaluation. c: E9.5 mouse embryos were dissected and placed in fixative at 4°C overnight. Total RNA was extracted and used for synthesis of 32P-labeled double-stranded cDNA. The cDNA was separated on an agarose gel, blotted to nylon filter, and visualized with a phosphoimager. d: Evaluation of the morphology of zinc-fixed 10-μm cryosections. Scale bar, 20 μm. n, neural tube; a, dorsal aorta.View Large Image Figure ViewerDownload Hi-res image Download (PPT)The effect of paraffin and OCT-embedding on cDNA synthesis was also evaluated. Frozen samples generated large amounts of long cDNA products, and losses during OCT embedding and freezing were small if any (Figure 2). Paraffin-embedded samples also generated fair amounts of cDNA, but not comparable to frozen samples (data not shown). Although tissue morphology was superior in paraffin sections, frozen sections were of sufficient quality (Figure 1d). Most importantly, the cells preserved their relative positions despite microscopic rifts.Figure 2Schematic picture of the experimental procedure and the estimated total RNA content in the sample at various stages.View Large Image Figure ViewerDownload Hi-res image Download (PPT)To summarize, zinc-fix efficiently recovers and preserves the integrity of RNA. Furthermore, zinc-fix can be performed prior to cryosectioning (contrary to precipitating agents), and was therefore chosen for all subsequent experiments.Experimental Procedure and RNA ContentAn experimental procedure for LMM and LPC isolation of cells from zinc-fixed frozen sections was worked out (Figure 2). Most of the steps are obvious necessities such as fixation, OCT embedding, cryosectioning, LMM/LPC, and RNA extraction. As LPC is most efficient with dry samples the sections were taken through a dehydration series before LMM. OCT interferes with laser cutting, and must be removed by 5 minutes of incubation in zinc-fix before dehydration. Total RNA content was measured at every step in the procedure to identify bottlenecks and to minimize losses (Figure 2). Generally, the RNA was stable when the cells were dry or frozen. RNA recovery was impaired at three steps—fixation, LMM/LPC, and RNA extraction. The evaluation of LMM/LPC and RNA extraction is discussed below. Fixation that reduces RNA recovery by ∼20% has already been covered.Laser microdissection with the PALM Robot-MicroBeam system is performed in two steps. First, the cells are outlined with a cutting laser in a process called LMM.14Schutze K Posl H Lahr G Laser micromanipulation systems as universal tools in cellular and molecular biology and in medicine.Cell Mol Biol. 1998; 44: 735-746PubMed Google Scholar Second, the cells are vertically transferred into a collecting device held in place above (no contact) the specimen in a process called LPC. The collector is coated with
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