Name: laser-capture microdissection of human prostatic epithelium for rna analysis
Uploaded: 2015-11-26T15:00:00
Description: Watch this Scientific Journal Video about Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis at JoVE.com

We thank Dr. Vicky Macias, Angeline Giangreco and Avani Vaishnav for assistance with optimizing this methodology over the years and Yang Zhang and Dr. Jian Ma at the University of Illinois at Urbana-Champaign for the RNA seq analysis. This work was supported by NIH/NCI R01 CA166588-01 (Nonn), American Cancer Society Research Scholar 124264-RSG-13-012-01-CNE (Nonn), NIH/NCI R03 CA172827-01 (Nonn), DOD-CDMPR PRCP Health Disparities Idea Award PC121923 (Nonn) and a Prevent Cancer Foundation grant (Zhou).

All human tissues used for these experiments were acquired via an Institutional Review Board approved protocol and/or exemption at University of Illinois at Chicago.
1. Section Fresh Frozen Prostate onto PEN-slide and onto Charged Glass Slide
<ol>
	<li>The day before or a few hours before sectioning the sample, clean tools (i.e., brush, forceps, coplins, blades, PEN-framed slides (if not already RNase free), an ETOH safe marker and a pencil) and the inside of a RT cryostat with RNase-decontaminating sol...

<ol>
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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=mRNA+and+micro-RNA+expression+analysis+in+laser-capture+microdissected+prostate+biopsies:+valuable+tool+for+risk+assessment+and+prevention+trials.">mRNA and micro-RNA expression analysis in laser-capture microdissected prostate biopsies: valuable tool for risk assessment and prevention trials.</a> Experimental and molecular pathology. 88, 45-51 (2010).</li><li>Long, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Global+transcriptome+analysis+of+formalin-fixed+prostate+cancer+specimens+identifies+biomarkers+of+disease+recurrence.">Global transcriptome analysis of formalin-fixed prostate cancer specimens identifies biomarkers of disease recurrence.</a> Cancer research. 74, 3228-3237 (2014).</li><li>Long, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein-coding+and+microRNA+biomarkers+of+recurrence+of+prostate+cancer+following+radical+prostatectomy.">Protein-coding and microRNA biomarkers of recurrence of prostate cancer following radical prostatectomy.</a> The American journal of pathology. 179, 46-54 (2011).</li><li>Giangreco, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+expression+and+regulation+of+vitamin+D+hydroxylases+and+inflammatory+genes+in+prostate+stroma+and+epithelium+by+1,25-dihydroxyvitamin+D+in+men+with+prostate+cancer+and+an+in+vitro.+model.">Differential expression and regulation of vitamin D hydroxylases and inflammatory genes in prostate stroma and epithelium by 1,25-dihydroxyvitamin D in men with prostate cancer and an in vitro. model.</a> The Journal of steroid biochemistry and molecular biology. , (2014).</li><li>Giangreco, A. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tumor+suppressor+microRNAs,+miR-100+and+-125b,+are+regulated+by+1,25-dihydroxyvitamin+D+in+primary+prostate+cells+and+in+patient+tissue.">Tumor suppressor microRNAs, miR-100 and -125b, are regulated by 1,25-dihydroxyvitamin D in primary prostate cells and in patient tissue.</a> Cancer prevention research. 6, 483-494 (2013).</li><li>Mihelich, B. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-183-96-182+cluster+is+overexpressed+in+prostate+tissue+and+regulates+zinc+homeostasis+in+prostate+cells.">miR-183-96-182 cluster is overexpressed in prostate tissue and regulates zinc homeostasis in prostate cells.</a> The Journal of biological chemistry. 286, 44503-44511 (2011).</li><li>Bevilacqua, C., Makhzami, S., Helbling, J. C., Defrenaix, P., Martin, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintaining+RNA+integrity+in+a+homogeneous+population+of+mammary+epithelial+cells+isolated+by+Laser+Capture+Microdissection.">Maintaining RNA integrity in a homogeneous population of mammary epithelial cells isolated by Laser Capture Microdissection.</a> BMC cell biology. 11, 95 (2010).</li><li>Gregg, J. L., Brown, K. E., Mintz, E. M., Piontkivska, H., Fraizer, G. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+gene+expression+in+prostate+cancer+epithelial+and+interstitial+stromal+cells+using+laser+capture+microdissection.">Analysis of gene expression in prostate cancer epithelial and interstitial stromal cells using laser capture microdissection.</a> BMC cancer. 10, 165 (2010).</li><li>Hart, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+microRNA+profiling+of+prostate+carcinomas+with+increasing+tumor+stage+by+deep+sequencing.">Comparative microRNA profiling of prostate carcinomas with increasing tumor stage by deep sequencing.</a> Molecular cancer research : MCR. 12, 250-263 (2014).</li><li>Smalheiser, N. R., Lugli, G., Thimmapuram, J., Cook, E. H., Larson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Endogenous+siRNAs+and+noncoding+RNA-derived+small+RNAs+are+expressed+in+adult+mouse+hippocampus+and+are+up-regulated+in+olfactory+discrimination+training.">Endogenous siRNAs and noncoding RNA-derived small RNAs are expressed in adult mouse hippocampus and are up-regulated in olfactory discrimination training.</a> Rna. 17, 166-181 (2011).</li><li>Song, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+profile+analysis+of+microRNAs+in+prostate+cancer+by+next-generation+sequencing.">Expression profile analysis of microRNAs in prostate cancer by next-generation sequencing.</a> The Prostate. 75, 500-516 (2015).</li><li>Deng, M. Y., Wang, H., Ward, G. B., Beckham, T. R., McKenna, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+six+RNA+extraction+methods+for+the+detection+of+classical+swine+fever+virus+by+real-time+and+conventional+reverse+transcription-PCR.">Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription-PCR.</a> Journal of veterinary diagnostic investigation : official publication of the American Association of Veterinary Laboratory Diagnosticians, Inc. 17, 574-578 (2005).</li><li>Kong, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+short+amplicons+in+FFPE-derived+long-chain+RNA.">Quantitative assessment of short amplicons in FFPE-derived long-chain RNA.</a> Scientific reports. 4, 7246 (2014).</li></ol>

Gene expression profiling from human specimens can be challenging, not only for the quality or quantity of tissue available, but also for the various histological entities present in a given tissue specimen. This is particularly challenging in the prostate in which benign tissues are largely stromal tissues and areas of cancer are devoid of stroma. LCM facilitates physical separation of prostatic stroma and epithelium RNA for a more accurate signature of the two different cell types (Figure 1A). In compa...

The prostate gland is a heterogeneous tissue composed of secretory epithelium arranged in glandular acini surrounded by fibromuscular stroma composed primarily of smooth muscle1. The epithelial compartment is comprised of five different but organized cell types: basal cells, secretory cells, neuroendocrine cells, transit amplifying cells and stem cells2. In prostate cancer (PCa), which arises from the luminal epithelial cells, the growth of the adenocarcinoma causes an evident progressive decline of the stromal compartment3. For these reasons, tissue specimens will have distinct differences in the proportion of stromal and epithelial cell type based on the extent of PCa. These differences can lead to biased assumptions of the gene expression data acquired from whole tissues without regard to microdissection of the desired cell type. Therefore, to remove this bias it is essential to separate cell types prior to RNA extraction and gene expression analysis.
Macrodissection or microdissection can be used to physically separate well-characterized epithelial areas from the surrounding stroma4-6. Macrodissection is typically done with a razor blade under a dissecting microscope and works well for separating large PCa nodules from stroma, but is not capable of removing benign epithelium from surrounding stroma (see example of benign prostate histology in Figure 1). Microdissection with a laser (LCM) is significantly more labor intensive than macrodissection, but can very accurately dissect benign epithelium4.
Recent publications from our lab have shown that RNA can be successfully extracted by LCM from either formalin-fixed paraffin-embedded (FFPE) biopsies or frozen tissue4,7-9. The major challenges in LCM-RNA extraction are 1) to accurately dissect the desired areas of the tissue, and 2) to preserve RNA integrity during the LCM and isolation process4,10. RNA isolated from pure cell populations can be used for gene expression analysis by several methods including reverse-transcription quantitative PCR (RT-qPCR)7,8, microarray11, and deep -sequencing12-14.
The goal of this protocol is to isolate total RNA from LCM prostatic epithelium from frozen tissue for downstream gene expression analyses.

<table>
	<tbody>
		<tr>
			<td>RNase-AWAY&#160;</td>
			<td>MBP</td>
			<td>7005-11</td>
			<td></td>
		</tr>
		<tr>
			<td>PEN-membrane 4,0 mm slides&#160;</td>
			<td>Leica</td>
			<td>11600289</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides, Superfrost Plus</td>
			<td>FisherBrand</td>
			<td>12-550-15</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 200 proof</td>
			<td>Decon labs.</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>DEPC (diethyl pyrocarbonate)</td>
			<td>Sigma</td>
			<td>D-5758</td>
			<td></td>
		</tr>
		<tr>
			<td>Cryostat</td>
			<td>Leica</td>
			<td>CM3050</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplins (Staining jar)</td>
			<td>IHCWORLD</td>
			<td>M900-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Coplins (Staining rack)</td>
			<td>IHCWORLD</td>
			<td>M905-12</td>
			<td></td>
		</tr>
		<tr>
			<td>Aperio ScanScope</td>
			<td>Aperio(Leica)</td>
			<td>ScanScope&#174; CS</td>
			<td></td>
		</tr>
		<tr>
			<td>Toluidine Blue</td>
			<td>Fluka</td>
			<td>89640-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>Laser Microdissection System</td>
			<td>Leica</td>
			<td>LMD7000</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL Thin-walled Tubes for LCM</td>
			<td>Thermo Scientific</td>
			<td>AB-0350</td>
			<td></td>
		</tr>
		<tr>
			<td>RNAqueous&#174;-Micro Total RNA Isolation Kit</td>
			<td>Ambion</td>
			<td>AM1931</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>NanoDrop</td>
			<td>Thermo Scientific</td>
			<td>ND-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit 2.0 Fluorometer</td>
			<td>Life Technologies</td>
			<td>Q32866</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>High-Capacity cDNA Reverse Transcription Kit</td>
			<td>Applied Biosystems</td>
			<td>4368814</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>Universal cDNA Synthesis Kit II, 8-64 rxns</td>
			<td>Exiqon</td>
			<td>203301</td>
			<td></td>
		</tr>
		<tr>
			<td>TaqMan microRNA RT kit</td>
			<td>Applied Biosystems</td>
			<td>4366597</td>
			<td>Thermo Fisher Scientific Brand</td>
		</tr>
		<tr>
			<td>Hematoxylin stain&#160;</td>
			<td>Ricca Chemical Company</td>
			<td>3536-16</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin-Y</td>
			<td>Richard Allan Scientific&#160;</td>
			<td>7111</td>
			<td></td>
		</tr>
	</tbody>
</table>

laser-capture microdissection of human prostatic epithelium for rna analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types. Healthy prostate is comprised of fibromuscular stroma surrounding discrete epithelial-lined secretory lumens and a very small population of immune and neuroendocrine cells. In contrast, areas of prostate cancer have increased dysplastic luminal epithelium with greatly reduced or absent stromal population. Given the profound difference between stromal and epithelial cell types, it is imperative to separate the cell types for any type of downstream molecular analysis. Despite this knowledge, the bulk of gene expression studies compare benign prostate to cancer without micro-dissection, leading to stromal bias in the benign samples. Laser-capture micro-dissection (LCM) is an effective method to physically separate different cell types from a specimen section. The goal of this protocol is to show that RNA can be successfully isolated from LCM-collected human prostatic epithelium and used for downstream gene expression studies such as RT-qPCR and RNAseq.

In a previous study we demonstrated the use of LCM to collect epithelial and stromal tissues to compare expression profiling by RT-qPCR of mRNA and microRNAs from frozen and FFPE prostate tissues from the same patient4. LCM is time consuming, particularly if large amount of RNA is to be collected for next-generation sequencing analysis. Therefore, it is crucial to keep the working space and tools RNase free. It is recommended to examine quality and cell-specificity controls in the RNA collected (discussed in m...

Watch this Scientific Journal Video about Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis at JoVE.com

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis (Video) | JoVE

The goal of this protocol is to use laser-capture micro-dissection as an effective method to isolate pure populations of cell types from heterogeneous prostate tissues for downstream RNA analysis.

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

The prostate gland contains a heterogeneous milieu of stromal, epithelial, neuroendocrine and immune cell types.  Healthy prostate is comprised of ...

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