Name: 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
Uploaded: 2011-11-13T14:00:00
Description: Watch this Scientific Journal Video about Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencin

This work was funded by the Defense Advanced Research Projects Agency (DARPA) and the Army Research Office (ARO), award number W911NF-10-1-006.

1. Sampling setup and preparation
<ol>
	<li>Supplement Earle's balanced salt solution (EBSS) with sodium bicarbonate (0.44 g per 100 mL EBSS) and glucose (0.884 g per 100 mL EBSS)9. Treat the EBSS with DEPC (0.1 mL per 100 mL EBSS) for at least 12 hours at 37 &deg;C in an autoclavable bottle or flask with a screw top.</li>
	<li>Place 5 &frac34;" glass Pasteur pipets into a 1000 mL glass beaker, and place artist's brushes, with bristles facing upward, into a 100 mL glass graduated cylinder. Add sufficient ...

<ol>
	<li>Pickrell, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Understanding+mechanisms+underlying+human+gene+expression+variation+with+RNA+sequencing.">Understanding mechanisms underlying human gene expression variation with RNA sequencing.</a> Nature. 464, 768-772 (2010).</li><li>Costa, V., Angelini, C., De Feis, I., Ciccodicola, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Uncovering+the+complexity+of+transcriptomes+with+RNA-Seq.">Uncovering the complexity of transcriptomes with RNA-Seq.</a> J. Biomed. Biotechnol. 853916, (2010).</li><li>Nagalakshmi, U., Waern, K., Snyder, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-Seq:+a+method+for+comprehensive+transcriptome+analysis.">RNA-Seq: a method for comprehensive transcriptome analysis.</a> Curr. Protoc. Mol. Biol. Chapter 4, 11-13 (2010).</li><li>Anglicheau, D., Muthukumar, T., Suthanthiran, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+small+RNAs+with+big+effects.">MicroRNAs: small RNAs with big effects.</a> Transplantation. 90, 105-112 (2010).</li><li>Forrest, A. R., Carninci, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+genome+transcriptome+analysis.">Whole genome transcriptome analysis.</a> RNA. Biol. 6, 107-112 (2009).</li><li>Twine, N. A., Janitz, K., Wilkins, M. R., Janitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Whole+transcriptome+sequencing+reveals+gene+expression+and+splicing+differences+in+brain+regions+affected+by+Alzheimer's+disease.">Whole transcriptome sequencing reveals gene expression and splicing differences in brain regions affected by Alzheimer's disease.</a> PLoS One. 6, e16266-e16266 (2011).</li><li>Paulsen, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gene+expression+profiling+of+individual+hypothalamic+nuclei+from+single+animals+using+laser+capture+microdissection+and+microarrays.">Gene expression profiling of individual hypothalamic nuclei from single animals using laser capture microdissection and microarrays.</a> J. Neurosci. Methods. 177, 87-93 (2009).</li><li>Winrow, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Refined+anatomical+isolation+of+functional+sleep+circuits+exhibits+distinctive+regional+and+circadian+gene+transcriptional+profiles.">Refined anatomical isolation of functional sleep circuits exhibits distinctive regional and circadian gene transcriptional profiles.</a> Brain. Res. 1271, 1-17 (2009).</li><li>Atkins, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+integration+of+glutamatergic+signals+by+little+SAAS+in+novel+suprachiasmatic+circuits.">Circadian integration of glutamatergic signals by little SAAS in novel suprachiasmatic circuits.</a> PLoS One. 5, e12612-e12612 (2010).</li><li>Technologies, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> MagMAX-96 Total RNA Isolation Kit Instruction Manual. , .</li></ol>

There are many critical steps in this protocol that warrant careful attention and consideration. Selection of the appropriate tissue corer diameter must be determined for removing each respective region of interest. While the tissue corer diameters presented in Table 1 are suitable for collection of the listed brain regions, each investigator should determine the appropriate-sized tissue corer for optimal collection of their respective tissue(s) of interest. Secondly, the coronal section thickness reported in this met...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Isotemp 102 water bath</td>
			<td>Fisher Scientific</td>
			<td>15-460-3Q</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNase Zap</td>
			<td>Ambion</td>
			<td>AM9782</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Lab rotator (orbital shaker)</td>
			<td>Thermo Scientific</td>
			<td>2314 &#x2013; 1CEQ</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Pellet mixer</td>
			<td>VWR</td>
			<td>47747-370</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>RNase-free 0.5 mL pestle</td>
			<td>VWR</td>
			<td>KT749521-0590</td>
			<td>Nuclease-free</td>
		</tr>
		<tr>
			<td>Diethyl pyrocarbonate (DEPC)</td>
			<td>Sigma Life Science</td>
			<td>D5758-50ML</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Double-edge razor blades</td>
			<td>Stoelting </td>
			<td>51427</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Stoelting tissue slicer with manual micrometer</td>
			<td>Stoelting </td>
			<td>51425</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>2/0 Round artist's brush</td>
			<td>Princeton Art &amp; Brush</td>
			<td>4350R</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Harris Uni-Core tissue corer (0.75 mm)</td>
			<td>Ted Pella</td>
			<td>15072</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Harris Uni-Core tissue corer (1.0 mm)</td>
			<td>Ted Pella</td>
			<td>15099</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Harris Uni-Core tissue corer (1.5 mm)</td>
			<td>Ted Pella</td>
			<td>15075</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Harris Uni-Core tissue corer (2.0 mm)</td>
			<td>Ted Pella</td>
			<td>15076</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Guillotine</td>
			<td>Braintree Scientific</td>
			<td>RG-100</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>DecapiCones</td>
			<td>Braintree Scientific</td>
			<td>MDC-200</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>HyClone Earle's balanced salt solution (EBSS)</td>
			<td>Thermo Scientific</td>
			<td>SH30029.02</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>25 mL pipets</td>
			<td>Corning</td>
			<td>4489</td>
			<td>Nuclease-free</td>
		</tr>
		<tr>
			<td>epT.I.P.S. LoRetention Dualfilter pipet tips, 20 &#x2013; 300 &mu;L</td>
			<td>Eppendorf</td>
			<td>0030 077.636</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>TrakMates screw top tubes with caps</td>
			<td>Thermo Scientific</td>
			<td>3741</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VisionMate wireless 2D barcode reader</td>
			<td>Thermo Scientific</td>
			<td>3122MTX</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>VisionMate SR single rack 2D barcode reader</td>
			<td>Thermo Scientific</td>
			<td>3115</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>1-200 &mu;L universal fit pipette tips</td>
			<td>Corning</td>
			<td>4864</td>
			<td>Nuclease-free</td>
		</tr>
		<tr>
			<td>Cellstar cell culture dishes</td>
			<td>Greiner Bio-One</td>
			<td>628160</td>
			<td>Nuclease-free</td>
		</tr>
		<tr>
			<td>Greiner Bio-One 96-well polystyrene Cellstar tissue culture plate, U-bottom</td>
			<td>USA Scientific</td>
			<td>5665-0180</td>
			<td>Nuclease-free</td>
		</tr>
		<tr>
			<td>#4 Filter paper</td>
			<td>Whatman</td>
			<td>1004 150</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Silicone bulb-type safety pipette filler</td>
			<td>Fisherbrand</td>
			<td>13-681-102A</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>NanoDrop 3300 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-3300</td>
			<td>&nbsp;</td>
		</tr>
	</tbody>
</table>

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.

Watch this Scientific Journal Video about Non-Laser Capture Microscopy Approach for the Microdissection of Discrete Mouse Brain Regions for Total RNA Isolation and Downstream Next-Generation Sequencin

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.

总RNA的分离和下游下一代测序和基因表达谱的非激光捕获显微切割离散小鼠脑地区显微镜方法

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

Research

JoVE Journal

Neuroscience

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

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

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

Micromanipulation of Gene Expression in the Adult Zebrafish Brain Using Cerebroventricular Microinjection of Morpholino Oligonucleotides

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Manipulation of gene expression in tissues is  required to perform functional studies. In this paper, we demonstrate the  cerebroventricular ...

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Developmental events in the brain including neuronal morphogenesis and migration are highly orchestrated processes. In vitro and in vivo analyses allow ...

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The molecular synaptic mechanisms underlying auditory learning and memory remain largely unknown. Here, the workflow of a proteomic study on auditory ...

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The ability to isolate specific brain regions of interest can be impeded in tissue disassociation techniques that do not preserve their spatial ...

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To gain a detailed understanding of the role of different CNS cells during development or the establishment and progression of brain pathologies, it is ...