Name: a stainless protocol for high quality rna isolation from laser capture microdissected purkinje cells in the human post-mortem cerebellum
Uploaded: 2019-01-17T14:45:00
Description: Watch this Scientific Journal Video about A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum at JoVE.com

The authors would like to acknowledge The New York Brain Bank, Dr. Jean Paul Vonsattel and Dr. Etty Cort&#233;s, for their assistance in cutting and preserving the frozen human brain tissue samples for these experiments. The authors would like to acknowledge the individuals who generously donated their brain for the continuing research into Essential Tremor. Dr. Faust and Dr. Martuscello would like to acknowledge Columbia University Department of Pathology and Cell Biology for their continued support and core research space. The authors would like to acknowledge NIH R01 NS088257 Louis/Faust) for research funding for this project. LCM images were performed in the Confocal and Specialized Microscopy Shared Resource of the Herbert Irving Comprehensive Cancer Center at Columbia University, supported by NIH grant #P30 CA013696 (National Cancer Institute). The authors would like to acknowledge Theresa Swayne and Laura Munteanu for their continued assistance with specialized microscopy.

All human samples utilized in this protocol have been obtained with informed consent and have been approved by the Internal Review Board (IRB) at Columbia University and Yale University.
NOTE: The entirety of this protocol should follow strict RNA handling guidelines, whereby a gloved hand is always used, all surfaces are cleaned with an RNase decontaminator and all working materials are RNA/DNA/Nuclease free.
1. Test RNA Integrity of Tissue prior to ...

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The protocol presented here is specifically modified to be a stainless approach in visualizing morphologically distinct tissues for UV-LCM. This method is designed to maximize RNA integrity for subsequent direct RNA sequencing, while maintaining an enhanced level of tissue visualization. The ability to distinguish different cell types for capture, to create the purest population of cells possible, is essential for understanding different molecular profiles in human tissues15. Within the context of...

Laser capture microdissection (LCM) is a valuable research tool that allows the separation of pathologically relevant cells for subsequent molecularly driven evaluation. The use of molecular analyses in these heterogeneous tissue specimens and the correlation with pathological and clinical data is a necessary step in evaluating the translational significance of biological research1. When analyzing gene expression data from RNA, the use of frozen tissue sections is highly recommended as it allows for excellent quality of RNA as well as maximized quantity2. It has been well established that high quality and quantity of RNA are essential for meaningful data from RNA sequencing3. However, when using RNA from fresh frozen post-mortem tissue for LCM, RNA degradation is a major challenge, as it occurs immediately upon death and its extent is mediated by various factors associated with the tissue collection method4,5. Furthermore, RNA degradation is exacerbated when staining techniques are needed to recognize histologic details and cell identification. Specialized staining techniques, such as hematoxylin &#38; eosin, Nissl stain, immunofluorescence and immunohistochemistry are helpful in differentiating cells from surrounding stroma but have been shown to degrade RNA and alter transcript expression profiles6. Therefore, our laboratory has created a stainless protocol specifically designed to preserve RNA in post-mortem human cerebellum for the purposes of RNA sequencing after LCM isolation of Purkinje neurons.
In processing fresh frozen tissue for LCM, the fixation method can variably affect both RNA and tissue integrity. Formalin fixation is standard for morphological preservation, but causes cross-linking that may fragment RNA and interfere with RNA amplification7. Ethanol fixation is a better alternative for RNA isolation, as it is a coagulative fixative that does not induce cross-linking1. To enhance the visualization of tissue morphology, xylene is the best choice, as it removes lipids from the tissue. However, there are known limitations when utilizing xylene in LCM, as the tissues can dry out and become brittle causing tissue fragmentation upon laser capture7. Xylene is also a volatile toxin and must be handled properly in a fume hood. Nevertheless, xylene has been shown to enhance tissue visualization while also preserving RNA integrity8. Therefore, our protocol centers around the use of 70% ethanol fixation and ethanol dehydration, followed by xylene incubation for morphological clarity.
It is important to note the different types of laser-based microdissection systems, as they have been shown to differ in speed, precision, and RNA quality. The infrared (IR) laser capture microdissection and the ultraviolet (UV) laser microbeam microdissection systems were both novel LCM platforms that emerged almost concurrently8. The IR-LCM system employs a &#34;contact system&#34; using a transparent thermoplastic film placed directly on the tissue section, and cells of interest selectively adhere to the film by focused pulses from an IR laser. Alternatively, the UV-LCM system is a &#34;non-contact system&#34; whereby a focused laser beam cuts away the cells or regions of interest in the tissue; depending on the configuration in two currently available commercial platforms that use either an inverted or upright microscope design, tissue is acquired into a collection device by a laser-induced pressure wave that catapults it against gravity or tissue is collected by gravity, respectively. Significant advantages of the UV-LCM system include faster cell acquisition, contamination-free collection with the non-contact approach and more precise dissection due to a much smaller laser beam diameter9. This protocol was specifically designed for a UV-LCM system and has not been tested in an IR-LCM system. In either UV-LCM system design, when accumulating cells into the collection cap, the use of a coverslip that enhances cellular clarity during microscopy is not suitable, as the cells would be unable to enter into the collection cap. Therefore, to enhance tissue visualization, we tested the use of Opaque collection caps, which are designed to act as a coverslip for microscopic visualization in UV-LCM systems, against liquid filled collection caps. Liquid filled collection caps can be challenging, as the liquid is subject to evaporation and must be replaced frequently while working at the microscope. Time becomes an important factor for RNA stability, as the captured tissue dissolves immediately7.
Our laboratory studies the postmortem neuropathologic changes in the cerebellum of patients with essential tremor (ET) and related neurodegenerative disorders of the cerebellum. We have demonstrated morphologic changes centered on Purkinje cells that distinguish ET cases versus controls, including a reduced number of Purkinje cells, increased dendritic regression and a variety of axonal changes, leading us to postulate that Purkinje cell degeneration is a core biologic feature in ET pathogenesis10,11,12,13. Transcriptional profiling has been used in many neurologic diseases to explore the underlying molecular basis of degenerative cellular changes. However, transcriptional profiles from heterogeneous samples, such as brain tissue regions, can effectually mask the expression of low abundance transcripts and/or diminish the detection of molecular changes that occur only in a small population of affected cells, such as Purkinje cells in the cerebellar cortex. For instance, Purkinje cells are vastly outnumbered by the abundant granule cells in the cerebellar cortex by approximately 1:3000; thus, to effectively target their transcriptome requires specific isolation of these neurons. The cerebellar cortex is delineated by distinct layers that differ in cell density and cell size. This cellular architecture is ideal to visualize in a fresh-frozen tissue sample without dye-containing staining reagents. In theory, this protocol could also be applied to other tissue types that have similar distinctive tissue organization.
This protocol was designed to work specifically for Purkinje cell visualization in the human post-mortem cerebellum. Numerous protocols exist for the fixation, staining visualization and RNA preservation of many types of tissues for the purposes of both IR- and UV-LCM. When contemplating an experimental design for UV-LCM, individuals should tailor their protocol to best fit the needs and requirements of starting and ending materials. Here, we combine many aspects of different LCM protocols to provide an enhanced method for visualizing Purkinje cells in the post-mortem human cerebellum without the need of dye containing staining reagents to prepare high-quality RNA for transcriptome sequencing.

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			<td height="42" style="height:42px;width:216px;">MembraneSlide NF 1.0 PEN</td>
			<td style="width:147px;">Zeiss</td>
			<td style="width:119px;">415190-9081-000</td>
			<td style="width:488px;">Membrane slides for tissue. We do not recommend using glass slides.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">AdhesiveCap 500 Opaque</td>
			<td style="width:147px;">Zeiss</td>
			<td style="width:119px;">415190-9201-000</td>
			<td style="width:488px;">Opaque caps are used to enhance visualization on inverted scopes only</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RNase Away</td>
			<td style="width:147px;">Molecular BioProducts</td>
			<td style="width:119px;">7005-11</td>
			<td style="width:488px;">Other RNase decontamination products are also suitable. Use to clean all surfaces prior to work.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">200 Proof Ethanol</td>
			<td style="width:147px;">Decon Laboratories</td>
			<td style="width:119px;">2701</td>
			<td style="width:488px;">If using alternative Ethanol, ensure high quality. Essential for tissue fixation.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Xylenes (Certified ACS)</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">X5P-1GAL</td>
			<td style="width:488px;">If using alternative xylene, ensure high quality. Essential for tissue visualization.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">UltraPure Distilled Water</td>
			<td style="width:147px;">Invitrogen</td>
			<td style="width:119px;">10977-015</td>
			<td style="width:488px;">Other RNA/DNA/Nuclease free water also suitable. Utilized in ethanol dilution only.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Slide-Fix Slide Jars</td>
			<td style="width:147px;">Evergreen</td>
			<td style="width:119px;">240-5440-G8K</td>
			<td style="width:488px;">Any slide holder/jar is also suitable. Must be cleaned prior to use. Used for fixation following sectioning.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-Roll Plate, Assy. 70mm 100um</td>
			<td style="width:147px;">Leica Biosystems</td>
			<td style="width:119px;">14041933980</td>
			<td style="width:488px;">Anti-roll plate type is determined by type of cryostat and attachment location on stage. All cryostats have differing requirements.&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Diethyl pyrocarbonate (DEPC)</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">D5758-50mL</td>
			<td style="width:488px;">DEPC from any vendor will do. Follow directions for water treatment.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Kimwipes</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">06-666A</td>
			<td style="width:488px;">Kimwipes can be ordered from any vendor and used at any size. Kimwipes are to be used to clean microscope and cryostat.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Tissue-Plus O.C.T. Compound</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">23-730-571</td>
			<td style="width:488px;">Any brand of OCT is acceptable. No tissue will be embedded in the OCT, it is strickly to attach tissue to &#39;chuck&#39;.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Edge-Rite Low-Profile Microtome Blades</td>
			<td style="width:147px;">Thermo Scientific</td>
			<td style="width:119px;">4280L</td>
			<td style="width:488px;">Blade type is determined by type of cryostat. All cryostats have differing requirements.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Leica Microsystems 3P&#160;25 + 30MM CRYOSTAT CHUCKS</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">NC0558768</td>
			<td style="width:488px;">Chuck type is determined by type of cryostat. All cryostats have different requirements. Either size is suitable.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RNeasy Micro Kit (50)</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:119px;">74004</td>
			<td style="width:488px;">Required for RNA extraction post LCM. RLT Lysis buffer essential to gather captured cells from opaque cap.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RNeasy Mini Kit (50)</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:119px;">74104</td>
			<td style="width:488px;">Required for RNA extraction of section preps, which are performed prior to LCM to test RNA integrity of starting tissues.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">RNase-Free DNase Set</td>
			<td style="width:147px;">Qiagen</td>
			<td style="width:119px;">79254</td>
			<td style="width:488px;">Dnase will remove any DNA to allow for a pure RNA population. Ensure Dnase is made fresh.&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">2-Mercaptoethanol</td>
			<td style="width:147px;">Sigma Aldrich</td>
			<td style="width:119px;">M6250-100ML</td>
			<td style="width:488px;">Purchased volume at user discretion. Necessary for addition to RLT buffer for cell lysis. Prevents RNase activity.&#160;</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Globe Scientific&#160;Lot Certified Graduated Microcentrifuge Tube (2 mL)</td>
			<td style="width:147px;">Fisher Scientific</td>
			<td style="width:119px;">22-010-092</td>
			<td style="width:488px;">Any 2 mL tube that is RNase free, DNase free, human DNA free, and Pyrogen free will do.&#160;</td>
		</tr>
	</tbody>
</table>

a stainless protocol for high quality rna isolation from laser capture microdissected purkinje cells in the human post-mortem cerebellum

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or regions from heterogenous tissues. Captured product can be used in a variety of molecular methods for protein, DNA or RNA isolation. However, preservation of RNA from postmortem human brain tissue is especially challenging. Standard visualization techniques for LCM require histologic or immunohistochemical staining procedures that can further degrade RNA. Therefore, we designed a stainless protocol for visualization in LCM with the intended purpose of preserving RNA integrity in post-mortem human brain tissue. The Purkinje cell of the cerebellum is a good candidate for stainless visualization, due to its size and characteristic location. The cerebellar cortex has distinct layers that differ in cell density, making them a good archetype to identify under high magnification microscopy. Purkinje cells are large neurons situated between the granule cell layer, which is a densely cellular network of small neurons, and the molecular layer, which is sparse in cell bodies. Because of this architecture, the use of stainless visualization is feasible. Other organ or cell systems that mimic this phenotype would also be suitable. The stainless protocol is designed to fix fresh-frozen tissue with ethanol and remove lipids with xylene for improved morphological visualization under high magnification light microscopy. This protocol does not account for other fixation methods and is specifically designed for fresh-frozen tissue samples captured using an ultraviolet (UV)-LCM system. Here, we present a full protocol for sectioning and fixing fresh frozen post-mortem human cerebellar tissue and purification of RNA from Purkinje cells isolated by UV-LCM, while preserving RNA quality for subsequent RNA-sequencing. In our hands, this protocol produces exceptional levels of cellular visualization without the need for staining reagents and yields RNA with high RNA integrity numbers (&#8805;8) as needed for transcriptional profiling experiments.

This protocol details the steps for preparing fresh frozen post-mortem human brain tissue for UV-LCM. Thorough specifications and annotations are given for cryostat cutting, as it can be difficult to cut fresh frozen brain tissue with a high degree of precision. The most important point to consider is that fresh frozen tissue is extremely cold and requires a significant amount of time to acclimate to the warmer temperature of a cryostat. This step cannot be rushed, and it cannot be overst...

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A Stainless Protocol for High Quality RNA Isolation from Laser Capture Microdissected Purkinje Cells in the Human Post-Mortem Cerebellum

This protocol uses a stain-free approach to visualize and isolate Purkinje cells in fresh-frozen tissue from human post-mortem cerebellum via laser capture microdissection. The purpose of this protocol is to generate sufficient amounts of high-quality RNA for RNA-sequencing.

Laser capture microdissection (LCM) is an advantageous tool that allows for the collection of cytologically and/or phenotypically relevant cells or ...

This protocol is significant, because it utilizes a stain-free visualization method instead of dye-based visualization on post-mortem human brain tissue, which helps preserve RNA integrity. The preservation of RNA is a big advantage in this protocol, as it is useful, not only in post-mortem human brain tissue, but also other tissue types, with high RNase activity. To begin, remove the tissue from the freezer and place it on dry ice for transport to the cryostat.Place the clean brushes, the chuck, and the tissue into the cryostat for at least 20 minutes, to allow them to reach the optimal temperature. For cryostats with dual chambers and specimen-holding temperatures, set the object temperature to minus 16 degrees Celsius and the chamber temperature to minus 18 degrees Celsius. Set the cryostat's section thickness to 14 micrometers and the trim thickness to 30 micrometers.Next, clean the stage with a one-to-one mixture of RNase decontaminator and 70%ethanol, to prevent freezing. Obtain a new, disposable cryostat blade and clean it with RNase decontaminator. Place the cleaned blade in the blade holder on the stage.Then, clean the anti-roll plate with the RNase decontaminator. Attach the cleaned plate to the stage and wait at least 20 minutes for the blade and anti-roll plate to come down to the temperature of the cryostat. First, cut a small piece of tissue for sectioning, making sure that the section is approximately 95%cerebellar cortex.Return the remaining tissue either to dry ice, or a to a freezer, at minus 80 degrees Celsius. Next, begin slowly adding OCT on top of the chuck with a slow, circular motion. Build up layers of OTC until there is a mount approximately three millimeters high covering the chuck.When the OCT is partially frozen, but still has some liquid in the center, place the tissues on top of the mount. Let the tissue sit on top of the OCT for one to two minutes until it is completely frozen. Then, transfer the chuck with the frozen OCT and tissue into the cryostat cutting arm.Adjust the tissue so that it is flush with the cutting blade and let the tissue sit in the cutting arm for 20 minutes to adjust to the new temperature. The most critical step is allowing the tissue to acclimate in the cryostat cutting arm for as long as necessary. If tissue shreds, purkinje cell visualization will be very difficult.I recommend using smaller, thinner pieces of tissue, to allow the tissue to acclimate faster. After this, slowly move the tissue closer to the blade. Once the tissue has reached the blade, begin the trimming process.Trim the tissue two to three times, until the cortex layers are visible. Next, place and align the anti-roll plate just above the cryostat blade. Cut four to six sections that are 14 micrometers thick, noting that properly cut sections will be flat under the anti-roll plate.Align the cut sections horizontally across the cryostat stage. Angle a slide to pick up all of the tissue pieces simultaneously. Immediately after sectioning the tissue, place the slide in the slide holder with 70%ethanol on ice for two minutes.Transfer the slide to a slide holder with 95%ethanol on ice for 45 seconds. Next, place the slide in the slide holder with 100%ethanol at room temperature for two minutes. Dip the slide three times into the slide holder containing xylene number one at room temperature.Then, place the slide in the slide holder with xylene two at room temperature for five minutes. Stand the slides up in a clean fume hood and let them air dry for at least 30 minutes. If storing the slides, place each one into a separate 50 milliliter tube and place the tubes into a freezer at minus 80 degrees Celsius for up to seven days.First, use RNase decontaminator to clean the microscope stage and the cap collection arm. With gloved hands, place the slide on the microscope and a 500 microliter opaque cap in the collection arm. At a low magnification, align the opaque cap over the cerebellar tissue, insuring that the cap covers the entire area that is visualized in the eye piece.Use a five times to 10 times objective to visualize the cerebellar layers. Place the cursor over the section where molecular layer and granual cell layer intersect. Move to a 40 times magnification and visualize the purkinje cells.Then, begin capturing them. To begin RNA collection after the microdissection, add 50 microliters of cell lysis buffer to the opaque cap with the cap facing up. Carefully close the tube over the cap and proceed with the collection as outlined in the text protocol.In this protocol, fresh, frozen, post-mortem, human brain tissue is prepared for UV laser capture microdissection. Following cryostat's sectioning in the allotted drawing time, the cellular layers of the cerebellum are easily visible with five times and 10 times objective lenses. As seen here, proper xylene incubation causes the tissue to become darker and better delineates cellular layers than does ethanol alone.When cutting at the laser capture microscope, the 40 times objective lens is required to ensure capturing only purkinje cells and not the surrounding tissue. As seen here, proper incubation in xylene produces a high quality morphologically intact image when compared to ethanol fixation alone. The cover slip ability of an opaque cap is then tested while other protocols utilize a liquid-filled cap, these caps can reduce tissue visualization and cause the resulting image to be granular and iridescent under the microscope.However, visualization through an opaque cap results in the smoothened tissue appearance that is softer and sharper in form. Representative images of excised purkinje cells at low power and at high power show precise removal of just the purkinje cell body. After this, high quality Rnase is obtained for subsequent RNA sequencing.Six representative samples underwent RNA extraction and were resuspended in 14 microliters of Rnase-free water and all six samples produced high quality RNA with RNA integrity numbers equal to or greater than eight. The most important thing to remember is that tissue quality is everything. Even cutting and slide placement will make or break this protocol.Following this procedure, any method that investigates RNA or DNA could be performed, specifically we probe our RNA for differential gene expression, but other questions regarding gene regulation could also be investigated. This method could be applied to any tissue type that has specific delineated morphology that does not require the use of antigen or dye-specific reagents for identification of cells of interest. We hope that this technique will help us understand the genetics of essential tremor and other diseases that have a tremor phenotype.The most hazardous chemical used in this protocol is xylene. It should be handled properly in a fume hood, disposed of properly, and slides should be allowed to dry adequately until used in an open space. Additionally, when working with human samples, biohazardous waste management, and safety should be utilized.

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Research

JoVE Journal

Neuroscience

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 ...

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of the breadth of molecular regulation. Newer approaches such as high-resolution RNA sequencing (RNA-Seq)1 provides new and expansive information about tissue- or state-specific expression such as relative transcript levels, alternative splicing, and micro RNAs2-4. Prospects for employing the RNA-Seq method in comparative whole transcriptome profiling5 within discrete tissues or between phenotypically distinct groups of individuals affords new avenues for elucidating molecular mechanisms involved in both normal and abnormal physiological states. Recently, whole transcriptome profiling has been performed on human brain tissue, identifying gene expression differences associated with disease progression6. However, the use of next-generation sequencing has yet to be more widely integrated into mammalian studies.
Gene expression studies in mouse models have reported distinct profiles within various brain nuclei using laser capture microscopy (LCM) for sample excision7,8. While LCM affords sample collection with single-cell and discrete brain region precision, the relatively low total RNA yields from the LCM approach can be prohibitive to RNA-Seq and other profiling approaches in mouse brain tissues and may require sub-optimal sample amplification steps. Here, a protocol is presented for microdissection and total RNA extraction from discrete mouse brain regions. Set-diameter tissue corers are used to isolate 13 tissues from 750-&mu;m serial coronal sections of an individual mouse brain. Tissue micropunch samples are immediately frozen and archived. Total RNA is obtained from the samples using magnetic bead-enabled total RNA isolation technology. Resulting RNA samples have adequate yield and quality for use in downstream expression profiling. This microdissection strategy provides a viable option to existing sample collection strategies for obtaining total RNA from discrete brain regions, opening possibilities for new gene expression discoveries.

Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencing and Gene Expression Profiling  |

RNA expression profiling of discrete mouse brain regions requires a precise and repeatable tissue collection strategy. A protocol that uses both coronal brain sectioning and tissue corer-assisted microdissection is described here. The yield and quality of total RNA obtained from the resulting samples confirms the utility of the outlined method.

As technological platforms, approaches such as next-generation sequencing, microarray, and qRT-PCR have great promise for expanding our understanding of ...

High-resolution Functional Magnetic Resonance Imaging Methods for Human Midbrain

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon measuring activity on the surface of cerebral cortex rather than in subcortical regions such as midbrain and brainstem. Subcortical fMRI must overcome two challenges: spatial resolution and physiological noise. Here we describe an optimized set of techniques developed to perform high-resolution fMRI in human SC, a structure on the dorsal surface of the midbrain; the methods can also be used to image other brainstem and subcortical structures.
High-resolution (1.2 mm voxels) fMRI of the SC requires a non-conventional approach. The desired spatial sampling is obtained using a multi-shot (interleaved) spiral acquisition1. Since, T2* of SC tissue is longer than in cortex, a correspondingly longer echo time (TE ~ 40 msec) is used to maximize functional contrast. To cover the full extent of the SC, 8-10 slices are obtained. For each session a structural anatomy with the same slice prescription as the fMRI is also obtained, which is used to align the functional data to a high-resolution reference volume.
In a separate session, for each subject, we create a high-resolution (0.7 mm sampling) reference volume using a T1-weighted sequence that gives good tissue contrast. In the reference volume, the midbrain region is segmented using the ITK-SNAP software application2. This segmentation is used to create a 3D surface representation of the midbrain that is both smooth and accurate3. The surface vertices and normals are used to create a map of depth from the midbrain surface within the tissue4.
Functional data is transformed into the coordinate system of the segmented reference volume. Depth associations of the voxels enable the averaging of fMRI time series data within specified depth ranges to improve signal quality. Data is rendered on the 3D surface for visualization.
In our lab we use this technique for measuring topographic maps of visual stimulation and covert and overt visual attention within the SC1. As an example, we demonstrate the topographic representation of polar angle to visual stimulation in SC.

This article describes techniques to perform high-resolution functional magnetic resonance imaging with 1.2 mm sampling in human midbrain and subcortical structures using a 3T scanner. Use of these techniques to resolve topographic maps of visual stimulation in the human superior colliculus (SC) is given as an example.

Functional MRI (fMRI) is a widely used tool for non-invasively measuring correlates of human brain activity. However, its use has mostly been focused upon ...

Isolation and Culture of Neural Crest Cells from Embryonic Murine Neural Tube

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to mesenchymal transition (EMT) and migrates throughout the embryo, giving rise to diverse cell types 1-3. NC also has the unique ability to influence the differentiation and maturation of target organs4-6. When explanted in vitro, NC progenitors undergo self-renewal, migrate and differentiate into a variety of tissue types including neurons, glia, smooth muscle cells, cartilage and bone. 
NC multipotency was first described from explants of the avian neural tube7-9. In vitro isolation of NC cells facilitates the study of NC dynamics including proliferation, migration, and multipotency. Further work in the avian and rat systems demonstrated that explanted NC cells retain their NC potential when transplanted back into the embryo10-13. Because these inherent cellular properties are preserved in explanted NC progenitors, the neural tube explant assay provides an attractive option for studying the NC in vitro. 
To attain a better understanding of the mammalian NC, many methods have been employed to isolate NC populations. NC-derived progenitors can be cultured from post-migratory locations in both the embryo and adult to study the dynamics of post-migratory NC progenitors11,14-20, however isolation of NC progenitors as they emigrate from the neural tube provides optimal preservation of NC cell potential and migratory properties13,21,22. Some protocols employ fluorescence activated cell sorting (FACS) to isolate a NC population enriched for particular progenitors11,13,14,17. However, when starting with early stage embryos, cell numbers adequate for analyses are difficult to obtain with FACS, complicating the isolation of early NC populations from individual embryos. Here, we describe an approach that does not rely on FACS and results in an approximately 96% pure NC population based on a Wnt1-Cre activated lineage reporter23. 
The method presented here is adapted from protocols optimized for the culture of rat NC11,13. The advantages of this protocol compared to previous methods are that 1) the cells are not grown on a feeder layer, 2) FACS is not required to obtain a relatively pure NC population, 3) premigratory NC cells are isolated and 4) results are easily quantified. Furthermore, this protocol can be used for isolation of NC from any mutant mouse model, facilitating the study of NC characteristics with different genetic manipulations. The limitation of this approach is that the NC is removed from the context of the embryo, which is known to influence the survival, migration and differentiation of the NC2,24-28.

Isolation of embryonic neural crest from the neural tube facilitates the use of in vitro methods for studying migration, self-renewal, and multipotency of neural crest.

The embryonic neural crest (NC) is a multipotent progenitor population that originates at the dorsal aspect of the neural tube, undergoes an epithelial to ...

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 ...

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or between the same cell type in health and disease or following pharmacologic treatments.&#160;In the spinal cord, such preparation from motor neurons, the target of interest in many neurologic and neurodegenerative diseases, is complicated by the fact that motor neurons represent &#60;10% of the total cell population.&#160;Laser capture microdissection (LMD) has been developed to address this problem.&#160;Here, we describe a protocol to quickly recover, freeze, and section mouse spinal cord to avoid RNA damage by endogenous and exogenous RNases, followed by staining with Azure B in 70% ethanol to identify the motor neurons while keeping endogenous RNase inhibited.&#160;LMD is then used to capture the stained neurons directly into guanidine thiocyanate lysis buffer, maintaining RNA integrity.&#160;Standard techniques are used to recover the total RNA and measure its integrity.&#160;This material can then be used for downstream analysis of the transcripts by RNA-seq and qRT-PCR.

High-quality total RNA has been prepared from cell bodies of mouse spinal cord motor neurons by laser capture microdissection after staining spinal cord sections with Azure B in 70% ethanol.&#160;Sufficient RNA (~40-60 ng) is recovered from 3,000-4,000 motor neurons to allow downstream RNA analysis by RNA-seq and qRT-PCR.

Preparation of high-quality RNA from cells of interest is critical to precise and meaningful analysis of transcriptional differences among cell types or ...

Feeder-free Derivation of Neural Crest Progenitor Cells from Human Pluripotent Stem Cells

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a source of transplantable cells for regenerative applications after disease or accidents. Neural crest (NC) cells are the precursors for a large variety of adult somatic cells, such as cells from the peripheral nervous system and glia, melanocytes and mesenchymal cells. They are a valuable source of cells to study aspects of human embryonic development, including cell fate specification and migration. Further differentiation of NC progenitor cells into terminally differentiated cell types offers the possibility to model human diseases in vitro, investigate disease mechanisms and generate cells for regenerative medicine. This article presents the adaptation of a currently available in vitro differentiation protocol for the derivation of NC cells from hPSCs. This new protocol requires 18 days of differentiation, is feeder-free, easily scalable and highly reproducible among human embryonic stem cell (hESC) lines as well as human induced pluripotent stem cell (hiPSC) lines. Both old and new protocols yield NC cells of equal identity.

Neural crest (NC) cells derived from human pluripotent stem cells (hPSC) have great potential for modeling human development and disease and for cell replacement therapies. Here, a feeder-free adaptation of the currently widely used in vitro differentiation protocol for the derivation of NC cells from hPSCs is presented.

Human pluripotent stem cells (hPSCs) have great potential for studying human embryonic development, for modeling human diseases in the dish and as a ...

Isolation of Distinct Cell Populations from the Developing Cerebellum by Microdissection

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. This method was first utilized in the study of Nestin-expressing progenitors (NEPs), a newly identified population of cells in the cerebellar external germinal layer (EGL). Using microdissection in combination with fluorescent-activated cell sorting (FACS), a pure population of NEPs was collected separately from conventional granule neuron precursors in the EGL and from other contaminating Nestin-expressing cells in the cerebellum. Without microdissection, functional analyses of NEPs would not have been possible with the current methods available, such as Percoll gradient centrifugation and laser capture microdissection. This technique can also be applied for use with various tissues that contain either recognizable regions or fluorescently-labeled cells. Most importantly, a major advantage of this microdissection technique is that isolated cells are living and can be cultured for further experimentation, which is currently not possible with other described methods.

Nestin-expressing&#160;progenitors are a newly identified population of neuronal progenitors in the developing cerebellum. Using the microdissection technique presented here in combination with fluorescent-activated cell sorting, this cell population can be purified with no contamination from other cerebellar regions and can be cultured for further studies.

Microdissection is a novel technique that can isolate specific regions of a tissue and eliminate contamination from cellular sources in adjacent areas. ...

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 ...

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the molecular and cellular basis of higher brain functions. A whole-mount preparation of adult brains with well-preserved morphology is critical for such whole brain-based studies, but can be technically challenging and time-consuming. This protocol describes an easy-to-learn, one-step dissection approach of an adult fly head in less than 10 s, while keeping the intact brain attached to the rest of the body to facilitate subsequent processing steps. The procedure helps remove most of the eye and tracheal tissues normally associated with the brain that can interfere with the later imaging step, and also places less demand on the quality of the dissecting forceps. Additionally, we describe a simple method that allows convenient flipping of the mounted brain samples on a coverslip, which is important for imaging both sides of the brains with similar signal intensity and quality. As an example of the protocol, we present an analysis of dopaminergic (DA) neurons in adult brains of WT (w1118) flies. The high efficacy of the dissection method makes it particularly useful for large-scale adult brain-based studies in Drosophila.

A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains

The adult Drosophila brain is a valuable system for studying neuronal circuitry, higher brain functions, and complex disorders. An efficient method to dissect whole brain tissue from the small fly head will facilitate brain-based studies. Here we describe a simple, one-step dissection protocol of adult brains with well-preserved morphology.

There is an increasing interest in using Drosophila to model human brain degenerative diseases, map neuronal circuitries in adult brains, and study the ...