This work is supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC: 418391-2012). AC receives a scholarship from the Centre th&#233;matique de recherche en Neurosciences (CTRN). HDB is funded by a scholarship from the Fonds de Recherche en Sant&#233; du Qu&#233;bec (FRSQ) and ML is a FRSQ Chercheur-Boursier.

NOTE: The experiments were performed in accordance with the Canadian Guide for the Care and Use of Laboratory Animals and were approved by the Universit&#233; Laval Animal Protection Committee.
1. Sample Preparation
NOTE: In this example, we used mouse brains at postnatal day 1.
<ol>
	<li>Use hypothermia anesthesia in crushed ice for 3 to 4 min for pups, then dissect brains as quickly as possible in ice-cold L15 medium. Perform this step within 2 min.</li>
	<li>Transfer dissected brains in embedding mold (cf. list of materiel) and fill with frozen tissue embedding media taking care to orient the specimen in the desired position. Freeze specimens immediately in liquid nitrogen and store at -80 &#176;C. Perform this step within 30 sec. 
	NOTE: Specimens can be stored few months without compromising the RNA quality.</li>
</ol>
2. Sectioning
<ol>
	<li>Treat membrane coated glass slides (cf. list of material) with surface RNAse decontamination solution to eliminate any trace of RNAses. Wash the slides in DEPC water and let them dry. Alternatively, bake slides overnight at 200 &#176;C.</li>
	<li>Transfer frozen samples in a cryostat previously cleaned with a surface RNAse decontamination solution. Set cryostat chamber temperature at -20 &#176;C and slice specimens at 10 &#181;m thickness. Collect tissue sections on the membrane coated slides and let them dry 10 min before staining. Alternatively, store slides at -80 &#176;C until staining.</li>
</ol>
3. Preparation of Staining Solutions
<ol>
	<li>Prepare all solutions just before the start of the experiment, and use RNAse inhibitor in all staining solutions (except for washing solutions) to prevent RNA degradation.</li>
	<li>Prepare the buffer solution. Use the same buffer in all solutions (composed of RNAse-free phosphate-buffered saline (PBS) supplemented of 1% BSA, 0.2% triton and 2% RNAse inhibitor). For each slide, prepare 400 &#181;l of buffer solution (200 &#181;l for the primary and 200 &#181;l for the secondary antibodies).</li>
	<li>Prepare the fixative solution: use 70% ethanol kept at -20 &#176;C. Keep tissue fixation procedure minimal to avoid a drastic reduction in the RNA extraction yield. 
	NOTE: Alternatively, use 95% ethanol solution supplemented of 5% acetic acid kept at -20 &#176;C.</li>
	<li>Prepare the primary antibody solution by diluting the primary antibody in the buffer solution. For dopaminergic neurons labeling, use an antibody targeting the tyrosine hydroxylase (TH, rate limiting enzyme for dopamine production). Use a high concentration of antibody to compensate for the quick incubation time. Use TH antibody at a dilution of 1:25. 
	NOTE: In normal immunofluorescence protocol, this antibody gives a strong signal when incubated overnight at a dilution of 1:1,000. In this protocol, it is used 40X more concentrated due to the short incubation time. Due to the high concentration of antibody used for this method, perform a standard immunostaining in parallel in order to check any increase in non-specific labeling.</li>
	<li>Prepare the secondary antibody solution by diluting the secondary antibody in the buffer solution. Use a biotinylated secondary antibody at a concentration of 1:100. 
	NOTE: In this example, we used an anti-rabbit biotinylated antibody.</li>
	<li>Prepare the avidin-biotin complex (ABC) solution for the detection of biotinylated secondary antibodies. Make ABC solution by adding the compound A and the compound B at a concentration of 1:100 diluted in DEPC PBS supplemented with 2% RNAse inhibitor. Prepare the Avidin-Biotin complex 30 min before adding it to the slides.</li>
</ol>
4. Staining
<ol>
	<li>Thaw one or two slides at the time from -80 &#176;C by flicking them in the air quickly (or use an air blower). Surround sections with a hydrophobic barrier pen and let dry for a few sec.
	<ol>
		<li>Put the slides in the cold fixative solution for no more than 5 min at -20 &#176;C. Shake once in the air to remove excess of fixative solution then, quickly dip slides in DEPC PBS 6-8 times for washing.</li>
		<li>Flick the slides to remove excess of DEPC PBS. Ensure that the slides defrosting step does not exceed 5 min.</li>
	</ol>
	</li>
	<li>Place slides on a clean tray (pretreated with a surface RNAse decontamination solution to eliminate any trace of RNAses) and put 200 &#181;l of the primary antibody solution on each slide (rabbit anti-TH) for 10 min at room temperature. Dip slides quickly in DEPC PBS 3 times for washing and flick the slides once to remove the excess of DEPC PBS.</li>
	<li>Put 200 &#181;l of the secondary antibody solution on each slide for 6 min at room temperature. Dip slides quickly 3 times in DEPC PBS for washing and flick the slides to remove the excess of DEPC PBS.</li>
	<li>Put 200 &#181;l of the ABC solution on each slide for 4 min at room temperature. Dip slides quickly in DEPC PBS 3 times for washing and flick the slides to remove the excess DEPC PBS.</li>
	<li>Prepare the DAB (3,3&#8217; diaminobenzidine) solution during the 4 min of incubation in the ABC solution. Use the reagents provided in the kit (DAB Peroxidase Substrate Kit). Mix 1 drop of provided buffer, 1 drop of DAB solution and 1 drop of 30% H2O2 in 2.5ml of DEPC water. Mix the solution by inverting.</li>
	<li>Take 196 &#181;l of the DAB solution and add 4 &#181;l (2%) of RNAse inhibitor solution just before spreading on slides. Place slides on a clean tray (pretreated with a surface RNAse decontamination solution) and put the 200 &#181;l of DAB solution on slides. 
	NOTE: DAB solution should react within1 or 2 min.</li>
	<li>Dip slides quickly in DEPC PBS 3 times for washing and put slides for few seconds in absolute ethanol. Dry the slides by flicking them in the air (or use an air blower). 
	NOTE: Slides can be stored at -80 &#176;C for future use or processed for LCM.</li>
</ol>
5. Laser Capture Microdissection
<ol>
	<li>Defrost slides quickly by flicking them in the air (or use an air blower). Ensure that the slides defrosting step does not exceed 5 min.</li>
	<li>Proceed to LCM. Place the slide under the microscope with the sample side facing the collecting tube cap. Choose the best magnification for the sample. Adjust the focus to see the cells of interest.
	<ol>
		<li>Define the cutting area and start cutting with the laser. Collect dissected cells or piece of tissue in the collecting tube cap filled with 50 &#181;l of lysis buffer (from RNA extraction kit). Ensure that the step of LCM does not exceed 30 min.</li>
	</ol>
	</li>
	<li>Extract total RNA from isolated cells using an RNA extraction kit (cf. list of materials). Follow the manufacturer&#8217;s protocol.
	<ol>
		<li>Incubate collected cells for 30 min at 42 &#176;C to lyse the cells. Pre-condition the RNA purification column by adding 250 &#956;l of conditioning buffer. Load 50 &#956;l of ethanol to lysed cells. Load the 100 &#956;l of lysed cells onto a preconditioned purification column.&#160;</li>
		<li>Centrifuge the column at 16,000 x g for 2 min at room temperature. Wash the column twice with washing buffers. Elute in the minimal recommended volume (11 &#181;l) of RNAse-free water (not DEPC treated as it may interfere with the bioanalyser). Keep 2 &#181;l to test quality. 
		NOTE: All solutions and reagents are provided in the kit.</li>
	</ol>
	</li>
</ol>
6. cDNA Synthesis and Quantitative RT-PCR
<ol>
	<li>Reverse-transcribe RNA samples from laser captured dopaminergic cells into complementary DNA using reverse transcriptase and oligo(dT). Follow the manufacturer&#8217;s protocol (cf. list of material).</li>
	<li>Use 1 &#181;l of cDNA for qRT-PCR amplification with gene-specific primers. Set up PCR reactions in 20 &#181;l volume with a one-component hot start reaction mix for quantitative PCR as recommended by the manufacturer. Carry out each reaction in triplicate.
	<ol>
		<li>For the experiments described here, use the following primer pairs: Gapdh-F(5&#8217;-CCA CCC AGA AGA CTG TGG AT-3&#8217;)/Gapdh-R (5&#8217;-GGA TGC AGG GAT GAT GTT CT-3&#8217;); Rpl13a-F (5&#8217;-ACA GCC ACT CTG GAG GAG AA-3&#8217;) / Rpl13a-R (5&#8217;-CTG CCT GTT TCC GTA ACC TC-3&#8217;) ; Th-F (5&#8217;-CAG TGG AGG ATG TGT CTC -3&#8217;) / Th-R (5&#8217;-GAA AAT CAC GGG CAG ACA G-3&#8217;); Aadc-F (5&#8217;- CAT GAG AGC TTC TGC CCT TC -3&#8217;)/Aadc-R (5&#8217;- GGA TGT GGT CCC CAG TGT AG -3&#8217;).</li>
	</ol>
	</li>
	<li>Perform quantitative RT-PCR amplification using the following rapid cycling parameters: 95 &#176;C for 5 min followed by 45 cycles of 10 sec 95 &#176;C, 10 sec at 60 &#176;C and 10 sec at 72 &#176;C. Perform melting curve analysis using the default setting of the instrument to determine homogeneous product formation.</li>
	<li>Analyze the data using the built-in software.
	<ol>
		<li>Normalize the expression values of DA markers genes Th and Aadc with the expression of the two reference genes Gapdh and Rpl13a to obtain relative expression levels as described previously14.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

The most critical point of this method consists in labeling cells of interest while preventing RNA degradation. As RNAses are active in aqueous solutions, decreasing incubation times improves RNA conservation11,15. All solutions used must be treated with DEPC to inactivate RNAses. All materials and surfaces need to be treated with a surface RNAse decontamination solution to prevent RNAses contamination. Finally, in these conditions, the addition of RNAse inhibitor in all solutions is effective in preserving RNA from being degraded. The use of high salt conditions applied during antibody incubation has been previously shown to be effective in preserving RNA9,10. However, this alternative method remains limited to quick staining using robust antibodies. The thickness of the sections represents another critical point and depends on the size of the cell of interest. Since the average size of the cell body of dopaminergic neurons is around 10&#181;m, we select this section thickness to obtain one layer of cells.
The major limitation of this technique compared to FACS is the low number of cells that can be microdissected. The use of RNA amplification kit is required for gene profiling experiments using RNA-sequencing or microarray16. However, qRT-PCR can be performed directly without the amplification step (Figure 2c). Qualitative assessment of the staining obtained with the quick immunolabeling protocol should always be performed and should not differ from the staining obtained using the normal immunolabeling protocol.
Until now, laser capture experiments were mostly used to isolate populations of cells from transgenic animals expressing markers like GFP or using quick histological colorations such as Nissl staining. The method presented here allows visualizing cells of interest using immunohistochemistry. Isolating and acquiring the gene expression profile of chemically identified populations of cells is now possible17. The protocol described here can be applied to any tissues and used with any suitable antibodies.
In the brain, neurons can be divided by their geographic localization but most brain regions contain a variety of different subpopulations based on their chemical identity. The gene expression pattern of certain types of neurons can be largely different from another cell type in the same cerebral area. According to the biological or pathological context, gene expression profile of a specific cell population can also change considerably. Using the procedure described here, it is possible to monitor these changes, paving the way to new discoveries8,18,19.

The brain is composed of a large variety of different neuron types forming complex networks. These neurons are organized in distinct groups and subgroups according to their morphology, connectivity and gene expression pattern1. The development of microarrays, next-generation sequencing, and qRT-PCR offered the possibility to compare gene expression profiles of neuronal populations in different biological contexts1,2. These sensitive analyses require the precise identification and isolation of cell types of interest while keeping RNA integrity2-4. Fluorescent activated cell sorting (FACS) technique has been widely used to isolate specific cell types based on cell-surface markers and/or morphology. FACS requires a cell dissociation step prior to sorting, which results in a complete loss of spatial resolution5. Many neuronal subpopulations are distinguished from one another according to their anatomical distribution in the brain. Laser capture microdissection applied on thin brain sections provides an appropriate option for specific isolation of cells with high spatial resolution6-8. A major limitation of LCM has been the need to identify cells of interest based on morphological criteria or on fluorescent protein reporters genetically engineered in animal models. The development of new techniques to perform quick immunolabeling methods that preserved RNA integrity, in combination with LCM, now allows the isolation of cell subpopulations to proceed with gene-profiling experiments.
This approach has been previously performed and briefly described in few publications1,9-13. Here, we demonstrate a detailed procedure to obtain high-quality RNA from a specific subset of cells in a complex tissue structure by combining quick immunolabeling with LCM. We show how to perform key critical steps to obtain maximal RNA recovery and avoid RNA degradation as it may significantly impact gene expression profiling.
For the demonstration of this protocol, dopaminergic neurons from one-day-old mouse midbrain were targeted. Dopaminergic neurons can be immunolabeled using an antibody directed against tyrosine hydroxylase (TH), the rate-limiting enzyme for dopamine synthesis. Following TH immunostaining, individual or groups of dopaminergic neurons can then be isolated using LCM. Microdissected cells are collected in the lysis buffer, and RNA is extracted using a RNA isolation kit. Quality and quantity of extracted RNA are measured using a bioanalyzer5. Further analysis of gene expression using: RNA sequencing, microarray, or qRT-PCR can subsequently be performed2,4,6. As an example, a two-step qRT-PCR is demonstrated on the laser captured isolated dopaminergic domains. Relative quantification of expression levels of two dopaminergic neuron marker genes illustrates the selectivity of this protocol.

<table><tbody><tr><td>Membrane coated slides&amp;nbsp;</td><td>Leica Biosystems</td><td>11505158</td><td>MembraneSlides PEN-Membrane 2,0 &amp;micro;m; 50 pcs</td></tr><tr><td>Surface RNAse decontamination solution</td><td>Life technologies</td><td>AM9780</td><td>RNaseZap</td></tr><tr><td>Fixative</td><td></td><td></td><td>70% Ethanol kept at -20 &amp;deg;C</td></tr><tr><td>Anti-TH&amp;nbsp;</td><td>Pel-Freez</td><td>P40101</td><td>&amp;nbsp;1:25</td></tr><tr><td>RNAse free buffer</td><td></td><td></td><td>DEPC PBS + 1% BSA+0.02% triton</td></tr><tr><td>RNAse inhibitor</td><td>Promega</td><td>N2615</td><td>Rnasine Plus Rnase Inhibitor 40 kU/ml (stock)</td></tr><tr><td>Anti-rabbit biotinylated</td><td>Vector Laboratories</td><td>BA-1000</td><td></td></tr><tr><td>Vectastain Elite ABC kit Standard</td><td>Vector Laboratories</td><td>PK-6100</td><td>8&amp;micro;l/ml</td></tr><tr><td>DAB Peroxidase Substrate Kit&amp;nbsp;</td><td>Vector Laboratories</td><td>SK-4100</td><td></td></tr><tr><td>RNA isolation kit</td><td>Life technologies</td><td>KIT0204</td><td>Arcturus PicoPure RNA Isolation Kit</td></tr><tr><td>H&lt;sub&gt;2&lt;/sub&gt;O&lt;sub&gt;2&lt;/sub&gt; 30%</td><td>Sigma</td><td>HC4060</td><td></td></tr><tr><td>Ethanol absolute</td><td></td><td></td><td></td></tr><tr><td>Laser microdissection system</td><td>Leica microsystems</td><td></td><td>Model AS-LMD</td></tr><tr><td>DEPC</td><td>Alfa Aesar</td><td>B22753-14</td><td></td></tr><tr><td>Bioanalyzer</td><td>Agilent</td><td></td><td>Agilent 2100 Bioanalyzer</td></tr><tr><td>Embedding mold&amp;nbsp;</td><td>Leica Biosystems</td><td>14702218311</td><td>6x8mm</td></tr><tr><td>Hydrophobic barrier pen&amp;nbsp;</td><td>Vector Laboratories</td><td>H-4000</td><td></td></tr><tr><td>Frozen tissue embedding media</td><td>Tissue-Tek</td><td>4583</td><td></td></tr><tr><td>Bioanalyzer chip</td><td>Aligent Technologies</td><td>5067-1513</td><td>RNA 6000 Pico LabChip used with Agilent 2100 Bioanalyzer</td></tr><tr><td>Hot start reaction mix for quantitative PCR</td><td>Roche</td><td>6402712001</td><td>Fast Start Essential DNA Green Master</td></tr><tr><td>Reverse transcriptase</td><td>Life technologies</td><td>11752-050</td><td>SuperScript III First-Strand Synthesis SuperMix for qRT-PCR</td></tr></tbody></table>

rna isolation from cell specific subpopulations using laser-capture microdissection combined with rapid immunolabeling

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

This rapid immunolabeling procedure allows visualization of cells of interest while keeping RNA integrity. Figure 1 illustrates the steps of tissue preparation before RNA extraction. After cryosectioning, dopaminergic neurons are labeled using an antibody directed against the tyrosine hydroxylase (labeled neurons appear in brown). Depending of the experimental question, single cells or larger region of interest can be isolated using LCM (Figure 1D-E). It is important to assess the quality of RNA extracted, as degraded RNA will have a considerable impact on the quality of the following analysis. Each sample is thus processed on a microfluidics-based platform bioanalyser for quantification and to check RNA integrity. Figure 2 shows typical bioanalyser results of degraded and good-quality samples. Both digital gel and electropherograms demonstrate the importance of using RNAse inhibitor in each solution to protect RNA. The expected amount of RNA extracted from 500 neurons is around 1-2 ng. Microdissected cells from several tissue sections can be pooled in the same tube to increase RNA quantity.
<img alt="Figure 1" src="/files/ftp_upload/52510/52510fig1.jpg" /> 
Figure 1: Rapid immunolabeling and laser capture microdissection procedure. (A) Schematic showing sagittal view of a mouse brain at embryonic day 15.5. Black line indicates the location of the anteroposterior regions where midbrain dopamine neurons are located. Thin sections are made using a cryostat and are collected on membrane coated glass slides. Following immunolabeling, midbrain dopaminergic neurons are visible in brown (B). Laser microdissected samples are collected in a tube cap filled with lysis buffer (C). Region of interest (blue line in D) or individual cell (E) can be dissected and retrieved in the tube cap. Scale bars: (D) 250&#956;m, (E) 50&#956;m. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52510/52510fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" src="/files/ftp_upload/52510/52510fig2.jpg" /> 
Figure 2: Quality control of total RNA sample isolated after laser capture microdissection using a bioanalyzer chip kit. (A) The digital gel obtained with the bioanalyzer (L, ladder; 1, sample 1 without RNAse inhibitor (RNAsine), sample 2 with RNAsine). (B) Electropherograms of sample 1 (upper panel) showing partially degraded RNA and sample 2 (lower panel) typical of good-quality RNA (RIN &#62;8 is acceptably good) in which the 18S/28S rRNA peaks are clearly visible. (C) Normalized relative expression levels represented in fold change for two DA marker genes (TH and AADC) between surrounding unstained tissue and DA domain. <a href="https://www-jove-com.bdigitaluss.remotexs.co/files/ftp_upload/52510/52510fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling at JoVE.com

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

rna-isolation-from-cell-specific-subpopulations-using-laser-capture

Nanyang Technological University

Research

JoVE Journal

Biology

12.4K Views.  Université Laval. Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

Video: RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Genetics

Laser Capture Microdissection of Drosophila Peripheral Neurons

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the molecular mechanisms underlying class-specific dendrite morphogenesis1,2. To facilitate molecular analyses of class-specific da neuron development, it is vital to obtain these cells in a pure population. Although a range of different cell, and tissue-specific RNA isolation techniques exist for Drosophila cells, including magnetic bead based cell purification3,4, Fluorescent Activated Cell Sorting (FACS)5-8, and RNA binding protein based strategies9, none of these methods can be readily utilized for isolating single or multiple class-specific Drosophila da neurons with a high degree of spatial precision. Laser Capture Microdissection (LCM) has emerged as an extremely powerful tool that can be used to isolate specific cell types from tissue sections with a high degree of spatial resolution and accuracy. RNA obtained from isolated cells can then be used for analyses including qRT-PCR and microarray expression profiling within a given cell type10-16. To date, LCM has not been widely applied in the analysis of Drosophila tissues and cells17,18, including da neurons at the third instar larval stage of development. 
Here we present our optimized protocol for isolation of Drosophila da neurons using the infrared (IR) class of LCM. This method allows for the capture of single, class-specific or multiple da neurons with high specificity and spatial resolution. Age-matched third instar larvae expressing a UAS-mCD8::GFP19 transgene under the control of either the class IV da neuron specific ppk-GAL420 driver or the pan-da neuron specific 21-7-GAL421 driver were used for these experiments. RNA obtained from the isolated da neurons is of very high quality and can be directly used for downstream applications, including qRT-PCR or microarray analyses. Furthermore, this LCM protocol can be readily adapted to capture other Drosophila cell types a various stages of development dependent upon the cell type specific, GAL4-driven expression pattern of GFP.

Laser Capture Microdissection of Drosophila Peripheral Neurons

In this video-article we present a method for isolating single or multiple Drosophila da neurons from third instar larvae using the infrared capture (IR) class of Laser Capture Microdissection (LCM). RNA obtained from the isolated neurons can be readily used for downstream applications including qRT-PCR or microarray analyses.

The dendritic arborization (da) neurons of the Drosophila peripheral nervous system (PNS) provide an excellent model system in which to investigate the ...

Neuroscience

Laser Capture Microdissection of Mammalian Tissue

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or formalin-fixed, paraffin-embedded tissue sections. LMD techniques rely on a thermo labile membrane placed either on top of, or underneath, the tissue section. In one method, focused laser energy is used to melt the membrane onto the underlying cells, which can then be lifted out of the tissue section. In the other, the laser energy vaporizes the foil along a path "drawn" on the tissue, allowing the selected cells to fall into a collection device. Each technique allows the selection of cells with a minimum resolution of several microns. DNA, RNA, protein, and lipid samples may be isolated and analyzed from micro-dissected samples. In this video, we demonstrate the use of the Leica AS-LMD laser microdissection instrument in seven segments, including an introduction to the principles of LMD, initializing the instrument for use, general considerations for sample preparation, mounting the specimen and setting up capture tubes, aligning the microscope, adjusting the capture controls, and capturing tissue specimens. Laser-capture micro-dissection enables the investigator to isolate samples of pure cell populations as small as a few cell-equivalents. This allows the analysis of cells of interest that are free of neighboring contaminants, which may confound experimental results.

Laser capture microscopy, also known as laser microdissection (LMD), enables the user to isolate small numbers of cells or tissues from frozen or ...

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that is critical for learning, memory and executive function.1,2 Hence our studies focus on gene expression analysis of specific neuronal populations in distinct subregions of the hippocampus. The technique of laser capture microdissection (LCM), introduced in 1996 by Emmert-Buck, et al.,3 has allowed for significant advances in gene expression analysis of single cells and enriched populations of cells from heterogeneous tissues such as the mammalian brain that contains thousands of functional cell types.4 We use LCM and a well established rat model of traumatic brain injury (TBI) to investigate the molecular mechanisms that underlie the pathogenesis of TBI. Following fluid-percussion TBI, brains are removed at pre-determined times post-injury, immediately frozen on dry ice, and prepared for sectioning in a cryostat. The rat brains can be embedded in OCT and sectioned immediately, or stored several months at -80 &deg;C before sectioning for laser capture microdissection. Additionally, we use LCM to study the effects of TBI on circadian rhythms. For this, we capture neurons from the suprachiasmatic nuclei that contain the master clock of the mammalian brain. Here, we demonstrate the use of LCM to obtain single identified neurons (injured and degenerating, Fluoro-Jade-positive, or uninjured, Fluoro-Jade-negative) and enriched populations of hippocampal neurons for subsequent gene expression analysis by real time PCR and/or whole-genome microarrays. These LCM-enabled studies have revealed that the selective vulnerability of anatomically distinct regions of the rat hippocampus are reflected in the different gene expression profiles of different populations of neurons obtained by LCM from these distinct regions. The results from our single-cell studies, where we compare the transcriptional profiles of dying and adjacent surviving hippocampal neurons, suggest the existence of a cell survival rheostat that regulates cell death and survival after TBI.

We describe how to use laser capture microdissection (LCM) to obtain enriched populations of hippocampal neurons or single neurons from frozen sections of the injured rat brain for subsequent gene expression analysis using quantitative real time PCR and/or whole-genome microarrays.

Long-term cognitive disability after TBI is associated with injury-induced neurodegeneration in the hippocampus-a region in the medial temporal lobe that ...

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor (EGF) and fibroblast growth factor (FGF) to provide an abundant supply of cells. NPCs were differentiated in the presence of a new combination of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), dibutyryl cAMP (DBC) and retinoic acid on dishes coated with poly-L-lysine and mouse laminin to obtain neuron-rich cultures. NPCs were also differentiated in the absence of neurotrophins, DBC and retinoic acid and in the presence of ciliary neurotrophic factor (CNTF) to yield astrocyte-rich cultures. Differentiated NPCs were characterized by immunofluorescence staining for a panel of neuronal markers including NeuN, synapsin, acetylcholinesterase, synaptophysin and GAP43. Glial fibrillary acidic protein (GFAP) and STAT3, astrocyte markers, were detected in 10-15% of differentiated NPCs. To facilitate cell-type specific molecular characterization, laser capture microdissection was performed to isolate neurons cultured on polyethylene naphthalate (PEN) membrane slides. The methods described in this study provide valuable tools to advance our understanding of the molecular mechanism of neurodegeneration.

Human neuroprogenitor cells (NPCs) were expanded under proliferating conditions. NPCs were differentiated into neuron-rich cultures in the presence of a combination of neurotrophins. Neuronal markers were detected by immunofluorescence staining. To isolate a pure population of neurons, NPCs were differentiated on PEN membrane slides and laser capture microdissection was performed.

Neuroprogenitor cells (NPCs) isolated from the human fetal brain were expanded under proliferative conditions in the presence of epidermal growth factor ...

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue sections on a microscope slide. When combined with immunohistochemistry, LCM can be used to isolate individual cells types based on a specific protein marker. Here, the LCM technique is described for collecting a specific population of dopamine neurons directly labeled with&#160;tyrosine hydroxylase immunohistochemistry and for isolation of the dopamine neuron containing region of the ventral tegmental area using indirect tyrosine hydroxylase immunohistochemistry on a section adjacent to those used for LCM. An infrared (IR) capture laser is used to both dissect individual neurons as well as the ventral tegmental area off glass slides and onto an LCM cap for analysis. Complete dehydration of the tissue with 100% ethanol and xylene is critical. The combination of the IR capture laser and the ultraviolet (UV) cutting laser is used to isolate individual dopamine neurons or the ventral tegmental area when using PEN membrane slides. A PEN membrane slide has significant advantages over a glass slide as it offers better consistency in capturing and collecting cells, is faster collecting large pieces of tissue, is less reliant on dehydration and results in complete removal of the tissue from the slide. Although removal of large areas of tissue from a glass slide is feasible, it is considerably more time consuming and frequently leaves some residual tissue behind. Data shown here demonstrate that RNA of sufficient quantity and quality can be obtained using these procedures for quantitative PCR measurements. Although RNA and DNA are the most commonly isolated molecules from tissue and cells collected with LCM, isolation and measurement of microRNA, protein and epigenetic changes in DNA can also benefit from the enhanced anatomical and cellular resolution obtained using LCM.

The isolation of individual dopamine neurons or the ventral tegmental area with direct or indirect immunohistochemistry is demonstrated using laser capture microdissection. Parameters for isolation of tissue from a glass slide using an infrared laser and from membrane slides using the combination of an infrared and ultraviolet laser are discussed.

Laser capture microdissection (LCM) is used to isolate a concentrated population of individual cells or precise anatomical regions of tissue from tissue ...

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, microvascular proliferation, mesenchymal transformation and necrosis characterize the histological heterogeneity of high-grade gliomas. Our laboratory has demonstrated that the presence of high densities of mesenchymal cells, named oncostreams, correlate with tumor malignancy. We have developed a unique approach to understand the mechanisms that underlie glioma&#39;s growth and invasion. Here, we describe a comprehensive protocol that utilizes laser capture microdissection (LMD) and RNA sequencing to analyze differential mRNA expression of intra-tumoral heterogeneous multicellular structures (i.e., mesenchymal areas or areas of tumor invasion). This method maintains good tissue histology and RNA integrity. Perfusion, freezing, embedding, sectioning, and staining were optimized to preserve morphology and obtain high-quality laser microdissection samples. The results indicate that perfusion of glioma bearing mice using 30% sucrose provides good morphology and RNA quality. In addition, staining tumor sections with 4% Cresyl violet and 0.5% eosin results in good nuclear and cellular staining, while preserving RNA integrity. The method described is sensitive and highly reproducible and it can be utilized to study tumor morphology in various tumor models. In summary, we describe a complete method to perform LMD that preserves morphology and RNA quality for sequencing to study the molecular features of heterogeneous multicellular structures within solid tumors.

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic analysis.

Gliomas are primary brain tumors characterized by their invasiveness and heterogeneity. Specific histological patterns such as pseudopalisades, ...

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene expression in these tissues. Thus, to analyze cell-specific gene expression profiles from Drosophila tissues, it may be necessary to isolate specific cell types with high purity and at sufficient yields for downstream applications such as transcriptional profiling and chromatin immunoprecipitation. However, the irregular cellular morphology in tissues such as the central nervous system, coupled with the rare population of specific cell types in these tissues, can pose challenges for traditional methods of cell isolation such as laser microdissection and fluorescence-activated cell sorting (FACS). Here, an alternative approach to characterizing cell-specific gene expression profiles using affinity-based isolation of tagged nuclei, rather than whole cells, is described. Nuclei in the specific cell type of interest are genetically labeled with a nuclear envelope-localized EGFP tag using the Gal4/UAS binary expression system. These EGFP-tagged nuclei can be isolated using antibodies against GFP that are coupled to magnetic beads. The approach described in this protocol enables consistent isolation of nuclei from specific cell types in the Drosophila larval central nervous system at high purity and at sufficient levels for expression analysis, even when these cell types comprise less than 2% of the total cell population in the tissue. This approach can be used to isolate nuclei from a wide variety of Drosophila embryonic and larval cell types using specific Gal4 drivers, and may be useful for isolating nuclei from cell types that are not suitable for FACS or laser microdissection.

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Drosophila tissues often contain a heterogeneous mixture of cell types.&#160;To examine gene expression in specific cell types from a particular tissue, nuclei can be genetically tagged and subsequently isolated using an affinity-based approach. Isolated nuclei can be used for downstream applications such as gene expression analysis and chromatin immunoprecipitation.

Drosophila melanogaster embryonic and larval tissues often contain a highly heterogeneous mixture of cell types, which can complicate the analysis of gene ...

Capturing Oral Submucous Fibrosis Tissue Samples for Spatial-Omics Analysis

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina propria and the submucosa are often found on histopathological slides. Epithelial dysplasia, epithelial atrophy, and senescent fibroblasts have been proposed to be associated with the malignant transformation of OSF. However, because of the heterogeneity of potentially malignant oral disorders and oral squamous cell carcinoma, it is difficult to identify the specific molecular mechanisms of malignant transformation in OSF. Here, we present a method to obtain a small number of epithelial or mesenchymal cells carrying morphological data and spatial information by laser capture microdissection on formalin-fixed paraffin-embedded tissue slides. Using a microscope, we can precisely capture microscale (~500 cells) dysplastic or atrophic epithelial tissue and fibrotic subepithelial tissue. The extracted cells can be evaluated by genome or transcriptome sequencing to acquire genomic and transcriptomic data with morphological and spatial information. This approach removes the heterogeneity of bulk OSF tissue sequencing and the interference caused by cells in non-lesioned areas, allowing for precise spatial-omics analysis of OSF tissue.

Author Spotlight: Unlocking the Mysteries of Oral Potential Malignancies 

Laser capture microdissection of oral submucous fibrosis tissues allows for precise extraction of cells from histological regions of interest for the analysis of multi-omics data with morphological and spatial information.

Oral submucous fibrosis (OSF) is a common type of potentially malignant disorder in the oral cavity. The atrophy of epithelium and fibrosis of the lamina ...

Medicine

Laser Microdissection for Species-Agnostic Single-Tissue Applications

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific genetic &#34;toolkits,&#34; such as promoters driving tissue-specific expression of fluorescent proteins. Further, protocols that disrupt tissues to isolate individual cells remove cells from their native environment (e.g., signaling from neighbors) and may result in stress responses or other differences from native gene expression states. In the present protocol, laser microdissection (LMD) is optimized to isolate individual nematode tail tips for the study of gene expression during male tail tip morphogenesis.
LMD allows the isolation of a portion of the animal without the need for cellular disruption or species-specific toolkits and is thus applicable to any species. Subsequently, single-cell RNA-seq library preparation protocols such as CEL-Seq2 can be applied to LMD-isolated single tissues and analyzed using standard pipelines, given that a well-annotated genome or transcriptome is available for the species. Such data can be used to establish how conserved or different the transcriptomes are that underlie the development of that tissue in different species.
Limitations include the ability to cut out the tissue of interest and the sample size. A power analysis shows that as few as 70 tail tips per condition are required for 80% power. Tight synchronization of development is needed to obtain this number of animals at the same developmental stage. Thus, a method to synchronize animals at 1 h intervals is also described.

A protocol is described that uses laser microdissection to isolate individual nematode tissues for RNA-sequencing. The protocol does not require species-specific genetic toolkits, allowing gene expression profiles to be compared between different species at the level of single-tissue samples.

Single-cell methodologies have revolutionized the analysis of the transcriptomes of specific cell types. However, they often require species-specific ...

RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling

Summary

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Chapters in this video

Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Laser Capture Microdissection of Drosophila Peripheral Neurons

Laser Capture Microdissection of Mammalian Tissue

Laser Capture Microdissection of Enriched Populations of Neurons or Single Neurons for Gene Expression Analysis After Traumatic Brain Injury

Laser Capture Microdissection of Neurons from Differentiated Human Neuroprogenitor Cells in Culture

Laser Capture Microdissection - A Demonstration of the Isolation of Individual Dopamine Neurons and the Entire Ventral Tegmental Area

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Affinity-based Isolation of Tagged Nuclei from Drosophila Tissues for Gene Expression Analysis

Isolation of Cells with Morphological and Spatial Information from Oral Submucous Fibrosis Samples by Laser Capture Microdissection

Laser Microdissection for Species-Agnostic Single-Tissue Applications