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 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 ...

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

Research

JoVE Journal

Biology

12.4K vistas.  Université Laval.  La expresión de genes de un subconjunto de células en un tejido específico representa un reto importante. En este artículo se describe cómo aislar el ARN total de alta calidad a partir de una población de células específicas mediante la combinación de un método immunolabeling rápida con láser captura microdissection. 

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

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 ...

RNA Extraction from Neuroprecursor Cells Using the Bio-Rad Total RNA Kit

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This methods paper describes how to isolate mid-to-late stage follicles (Stage 10B-14) and utilize them to provide new insights into the molecular and morphologic events occurring during tight windows of developmental time. Isolated follicles can be used for a variety of experimental techniques, including in vitro development assays, live imaging, mRNA expression analysis and western blot analysis of proteins. Follicles at Stage 10B (S10B) or later will complete development in culture; this allows one to combine genetic or pharmacologic perturbations with in vitro development to define the effects of such manipulations on the processes occurring during specific periods of development. Additionally, because these follicles develop in culture, they are ideally suited for live imaging studies, which often reveal new mechanisms that mediate morphological events. Isolated follicles can also be used for molecular analyses. For example, changes in gene expression that result from genetic perturbations can be defined for specific developmental windows. Additionally, protein level, stability, and/or posttranslational modification state during a particular stage of follicle development can be examined through western blot analyses. Thus, stage-specific isolation of Drosophila follicles provides a rich source of information into widely conserved processes of development and morphogenesis.

The Utility of Stage-specific Mid-to-late Drosophila Follicle Isolation

Stage-specific isolation of mid-to-late Drosophila follicles is useful for a variety of purposes. Such follicles develop in culture, which allows for genetic and/or pharmacologic manipulations to be coupled with in vitro development assays and live imaging. Additionally, follicles can be used for molecular studies, such as isolating mRNA and protein.

Drosophila oogenesis or follicle development has been widely used to advance the understanding of complex developmental and cell biologic processes. This ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Laser Capture Microdissection of Highly Pure Trabecular Meshwork from Mouse Eyes for Gene Expression Analysis

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great tool for the study of the molecular mechanisms underlying cell differentiation and the development and progression of various diseases, including glaucoma. Glaucoma, which comprises a family of progressive optic neuropathies, is the most common cause of irreversible blindness worldwide. Structural changes and damage within the trabecular meshwork (TM) can result in increased intraocular pressure (IOP), which is a major risk factor for developing glaucoma. However, the precise molecular mechanisms involved are still poorly understood. The ability to perform gene expression analysis will be crucial in obtaining further insights into the function of these cells and its role in the regulation of IOP and glaucoma development. To achieve this, a reproducible method for isolating highly enriched TM from frozen sections of mouse eyes and a method for downstream gene expression analysis, such as RT-qPCR and RNA-Seq is needed. The method described herein is developed to isolate highly pure TM from mouse eyes for downstream digital PCR and microarray analysis. In addition, this technique can be easily adapted for the isolation of other highly enriched ocular cells and cell compartments that have been difficult to isolate from mouse eyes. The combination of LCM and RNA analysis can contribute to a more comprehensive understanding of the cellular events underlying glaucoma.

Here, we describe a protocol for a reproducible laser capture microdissection (LCM) for isolating trabecular meshwork (TM) for downstream RNA analysis. The ability to analyze changes in gene expression in the TM will help in understanding the underlying molecular mechanisms of TM-related ocular diseases.

Laser capture microdissection (LCM) has allowed gene expression analysis of single cells and enriched cell populations in tissue sections. LCM is a great ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

Rapid Lipid Droplet Isolation Protocol Using a Well-established Organelle Isolation Kit

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral lipids encased by a monolayer of phospholipids and proteins. Hepatic LD lipids, such as ceramides, and proteins are implicated in several diseases that cause hepatic steatosis. Although previous methods have been established for LD isolation, they require a time-consuming preparation of reagents and are not designed for the isolation of multiple subcellular compartments. We sought to establish a new protocol to enable the isolation of LDs, endoplasmic reticulum (ER), and lysosomes from a single mouse liver.
Further, all reagents used in the protocol presented here are commercially available and require minimal reagent preparation without sacrificing LD purity. Here we present data comparing this new protocol to a standard sucrose gradient protocol, demonstrating comparable purity, morphology, and yield. Additionally, we can isolate ER and lysosomes using the same sample, providing detailed insight into the formation and intracellular flux of lipids and their associated proteins.

This protocol establishes a novel method of lipid droplet isolation and purification from mouse livers, using a well-established endoplasmic reticulum isolation kit.

Lipid droplets (LDs) are bioactive organelles found within the cytosol of the most eukaryotic and some prokaryotic cells. LDs are composed of neutral ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...