Name: rna isolation from cell specific subpopulations using laser-capture microdissection combined with rapid immunolabeling
Uploaded: 2015-04-11T14:00:00
Description: Watch this Scientific Journal Video about RNA Isolation from Cell Specific Subpopulations Using Laser-capture Microdissection Combined with Rapid Immunolabeling at JoVE.com

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>Tr...

<ol>
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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 RN...

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 border="0" cellpadding="0" cellspacing="0" width="895">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Name of Material/ Equipment</td>
			<td style="width:148px;">Company</td>
			<td style="width:99px;">Catalog Number</td>
			<td style="width:420px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Membrane coated slides&#160;</td>
			<td style="width:148px;">Leica Biosystems</td>
			<td style="width:99px;">11505158</td>
			<td>MembraneSlides PEN-Membrane 2,0 &#181;m; 50 pcs</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Surface RNAse decontamination solution</td>
			<td style="width:148px;">Life technologies</td>
			<td style="width:99px;">AM9780</td>
			<td>RNaseZap</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Fixative</td>
			<td style="width:148px;"></td>
			<td style="width:99px;"></td>
			<td>70% Ethanol kept at -20 &#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Anti-TH&#160;</td>
			<td style="width:148px;">Pel-Freez</td>
			<td style="width:99px;">P40101</td>
			<td>&#160;1:25</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">RNAse free buffer</td>
			<td style="width:148px;"></td>
			<td style="width:99px;"></td>
			<td>DEPC PBS + 1% BSA+0.02% triton</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">RNAse inhibitor</td>
			<td style="width:148px;">Promega</td>
			<td style="width:99px;">N2615</td>
			<td>Rnasine Plus Rnase Inhibitor 40 kU/ml (stock)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Anti-rabbit biotinylated</td>
			<td style="width:148px;">Vector Laboratories</td>
			<td style="width:99px;">BA-1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Vectastain Elite ABC kit Standard</td>
			<td style="width:148px;">Vector Laboratories</td>
			<td style="width:99px;">PK-6100</td>
			<td>8&#181;l/ml</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">DAB Peroxidase Substrate Kit&#160;</td>
			<td style="width:148px;">Vector Laboratories</td>
			<td style="width:99px;">SK-4100</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">RNA isolation kit</td>
			<td style="width:148px;">Life technologies</td>
			<td style="width:99px;">KIT0204</td>
			<td>Arcturus PicoPure RNA Isolation Kit</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">H2O2 30%</td>
			<td style="width:148px;">Sigma</td>
			<td style="width:99px;">HC4060</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Ethanol absolute</td>
			<td style="width:148px;"></td>
			<td style="width:99px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Laser microdissection system</td>
			<td style="width:148px;">Leica microsystems</td>
			<td style="width:99px;"></td>
			<td>Model AS-LMD</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">DEPC</td>
			<td style="width:148px;">Alfa Aesar</td>
			<td style="width:99px;">B22753-14</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Bioanalyzer</td>
			<td style="width:148px;">Agilent</td>
			<td style="width:99px;"></td>
			<td>Agilent 2100 Bioanalyzer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Embedding mold&#160;</td>
			<td style="width:148px;">Leica Biosystems</td>
			<td style="width:99px;">14702218311</td>
			<td>6x8mm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Hydrophobic barrier pen&#160;</td>
			<td style="width:148px;">Vector Laboratories</td>
			<td style="width:99px;">H-4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Frozen tissue embedding media</td>
			<td style="width:148px;">Tissue-Tek</td>
			<td style="width:99px;">4583</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Bioanalyzer chip</td>
			<td style="width:148px;">Aligent Technologies</td>
			<td style="width:99px;">5067-1513</td>
			<td>RNA 6000 Pico LabChip used with Agilent 2100 Bioanalyzer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:228px;">Hot start reaction mix for quantitative PCR</td>
			<td style="width:148px;">Roche</td>
			<td style="width:99px;">6402712001</td>
			<td>Fast Start Essential DNA Green Master</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:228px;">Reverse transcriptase</td>
			<td style="width:148px;">Life technologies</td>
			<td style="width:99px;">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 ...

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 ...

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