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 solution using a spray bottle and lab-wipes.
	<ol>
		<li>Allow RNase-decontaminating solution to sit for 5 min before wiping away, rinse with depcH20 to remove left over RNase-decontaminating solution and a final rinse with 70% EtOH (prepared with depcH20). 
		Note: It is essential to maintain an RNase-free workspace. All coplins, tools, frozen mounting medium, blades and slides used should be used only for RNase free work and never used in a standard non-RNase free environment. All instruments must be treated with an RNase decontamination solution before each experiment.</li>
	</ol>
	</li>
	<li>Cool cryostat to -24 &#176;C.</li>
	<li>Place the following cleaned RNase free instruments inside cryostat to cool at least 30 min prior to sectioning: slide coplin-filled with 100% ethanol, tools, small frozen tissue cassettes and mounting chuck.</li>
	<li>Retrieve frozen prostate tissue from cryostorage and place into a dry-ice-filled foam bucket for transport. Place tissue into cryostat to equilibrate to cryostat temperature for at least 30 min. 
	Note: Human prostate tissues for laser-capture micro-dissection can be either fresh-frozen or formalin-fixed paraffin-embedded (FFPE). The time constraints and slide preparation are slightly different between tissue sources. Here we describe the steps for frozen sections.</li>
	<li>Work with one tissue at a time, and squirt frozen mounting medium into the bottom of the cassette and quickly mount the frozen prostate tissue into the frozen mounting medium using the chilled forceps.</li>
	<li>Place the cassette with tissue onto chiller in cryostat for 5 min to set frozen mounting medium.</li>
	<li>Squirt a small amount of frozen mounting medium onto the chuck and carefully press mounted tissue bottom side up onto the frozen mounting medium. Let set in the cryostat for 5 min to allow the frozen mounting medium to completely solidify.</li>
	<li>Place chuck into the holder and tighten.</li>
	<li>Lock the handle in locked position and place a new blade onto the cryostat. For downstream molecular endpoints that include an amplification step (i.e.,&#160;qPCR) it is advisable to use a new blade for each patient to prevent contamination (or move the blade over to use area of the blade for the next sample).</li>
	<li>Unlock the handle and carefully advance the tissue by hand or with a foot pedal to even and expose the tissue with 50&#160;&#181;m sections until the desired tissue exposure is reached.</li>
	<li>Adjust cryostat to 5 &#181;m, cut one or two sections and place them onto charged glass slide for hematoxylin and eosin (H&#38;E) staining (see Step 2).</li>
	<li>Immediately place the slide into the cold 100% ethanol for 2 min.</li>
	<li>Remove the charged glass slide from ethanol and let it dry at RT before proceeding to H&#38;E staining (Step 2).</li>
	<li>Place RT PEN-frame slide on a frame supporter.</li>
	<li>Adjust cryostat to 10 &#181;m and place sections onto the RT PEN frame slides. One to four sections can be place onto each slide depending on the tissue size.</li>
	<li>Immediately plunge the slide into the cold 100% ethanol for 2 min.</li>
	<li>Remove the PEN-frame slides from ethanol and store the slide in a slide box in a desiccator box in a -80 &#176;C freezer until ready for LCM. Three days is the maximum storage time for optimal RNA recovery.</li>
	<li>Proceed with Step 2 for ONLY the charged glass slide. Proceed with Step 3 for PEN-frame slide.</li>
</ol>
2. Hematoxylin and Eosin-Y (H&#38;E) Stain for Histological Mark-up
Note: This if for the tissue on the charged glass slide and NOT the PEN-frame slide for LCM.
<ol>
	<li>Hydrate the sections on the charged glass slide dipping the slide through graded ethanol as follows: dip the slide two times in 100% ethanol for 3 min, two times in 95% ethanol for 3 min, once in 70% ethanol for 3 min and two times in distilled H2O for 3 min.
	<ol>
		<li>Prepare acid alcohol solution: 99 ml 70% ethanol + 1 ml 1% HCl and 0.1% sodium bicarbonate solution: 0.1 g Sodium bicarbonate in 100 ml distilled H2O</li>
	</ol>
	</li>
	<li>Dip the slide in Hematoxylin Gill II (0.4%) for 5 min.</li>
	<li>Wash off excess stain in steady running tap H2O for 3 min.</li>
	<li>Dip the slide 10 times in acid alcohol</li>
	<li>Wash the slide in running tap H2O for 3 min.</li>
	<li>Dip the slide in 0.1% sodium bicarbonate solution for 30-60 sec to turn the purplish color to blue.</li>
	<li>Wash the slide in running tap H2O for 3 min.</li>
	<li>Rinse the slide in 95% ethanol with 10 dips.</li>
	<li>Dip the slide in Eosin-Y 2-3 times for 15 sec total.</li>
	<li>Dehydrate the slide through graded ethanol as follows: dip the slide two times in 95% ethanol, two times in 100% ethanol, and two times in Xylene (make sure the tissue appears clear which indicates thorough dehydration).</li>
	<li>Mount the slide with non-aqueous permanent hard mounting medium and cover slip.</li>
	<li>Let the slide dry for 30 min.</li>
	<li>Scan slide with any whole slide scanner and print a large, high-resolution image onto 8&#8221; x 11&#8221; paper.</li>
	<li>Outline areas of interest (typically done by a board certified pathologist) with a marker. This markup is the guide for the LCM procedure.</li>
</ol>
3. Toluidine Blue Staining of PEN-frame Slides
Note: This is done the SAME DAY as the LCM. Use depcH2O water for all solutions. Complete all steps in RNase-free area. All coplins must be cleaned with RNase-decontaminating solution.
<ol>
	<li>The day of LCM remove the PEN-frame slides from -80 &#176;C freezer and hydrate by gently dipping slides into 90% EtOH for 2 min then 75% EtOH for 2 min.
	<ol>
		<li>Prepare 0.5% toluidine blue by dissolving in molecular biology grade water then filter sterilize through a 0.2 &#181;m filter and store at RT. This solution may be re-used for up to 6 months. Note: It is highly advised to prepare the solution prior to as the filtration can take a few hr.</li>
	</ol>
	</li>
	<li>Stain the prostate tissue by gently dipping slides into 0.5% toluidine blue for 5-30 sec. Destain the tissue by washing slides 2 times in depcH2O for 15 sec followed by 75% EtOH for 30 sec to 3 min. Note: stain and destain times can be adjusted to ensure good staining. Prostate tends to over stain.</li>
	<li>Transfer PEN-frame slides to RNAase-free dry container on ice.</li>
</ol>
4. Laser-capture Micro-dissection (LCM) &#160;
<ol>
	<li>Immediately prior to LCM, dry slide on a slide warmer at 42 &#176;C for 15 min. Dry only the slide that will be processed and store the rest on ice until ready for them.</li>
	<li>Turn on LCM and computer in this order, microscope power, the laser key, the laser switch and then the computer. Launch the Laser Microdissection software.</li>
	<li>Use the software to control the microscope to load the slides and collection tubes. Click on &#8220;unload sample holder&#8221;, load the slide with the sections onto the microscope slide holder with the tissue facing down and insert the slide holder in the appropriate slot in the microscope, then click &#8220;continue&#8221;.
	<ol>
		<li>Click on &#8220;unload collection tubes&#8221;, label and load 0.5 ml tubes onto tube holder and insert it in the appropriate slot in the instrument. Once caps are secured, add 35 &#956;l of Lysis buffer to the collection caps and try to avoid bubbles. Then click &#8220;OK&#8221;. 
		Note: If evaporation is an issue, dilute the buffer as follows: 25 &#181;l lysis buffer with 10 &#181;l depcH2O.</li>
	</ol>
	</li>
	<li>In the software, select the position where the slide has been placed in the slide holder. Select the collection cap to collect the LCM specimen.</li>
	<li>Visualize and focus the slide through the computer at 10X. Use the software to calibrate the laser by selecting &#8220;Laser&#8221; then &#8220;calibrate&#8221;. Adjust the power, aperture and speed of the laser, selecting &#8220;laser&#8221; then &#8220;control&#8221; to allow the laser beam to cut the area selected completely and efficiently. Repeat adjustments until the laser cuts completely through the tissue.</li>
	<li>Using the 8&#8221; x 11&#8221; pathologist mark-up as a guide, use the &#8220;draw shape&#8221; tool to circumscribe the desired areas of epithelium in the view and avoid collecting any stroma. 
	Note: Multiple areas can be selected within one view, but do not move to another view without first cutting by clicking the &#8220;cut shape&#8221; tool.
	<ol>
		<li>If an area does not get completely cut by the laser use the &#8220;move and cut&#8221; tool to go over it manually. Continue moving the objective to new views and repeat laser cutting until slide is completely dissected or the desired amount of tissue has been collected (typically 100 benign glands yield 150-500 ng of RNA). (see example in Figure 1A).</li>
	</ol>
	</li>
	<li>Carefully remove tubes from the collection holder and close the caps, being mindful of the lysis buffer and tissue in the caps. Briefly centrifuge for 30 sec to collect liquid and tissue in the bottom of the tube. Incubate the samples at 42 &#176;C for 30 min and store at -80 &#176;C until ready for RNA isolation.</li>
</ol>
5. Total RNA Isolation with a Filter-based Kit and Quality Analysis
<ol>
	<li>Thaw out the tubes on ice and bring the volume of the solution to 100 &#181;l.</li>
	<li>Pre-wet the micro filter of an RNA isolation kit by applying 30 &#181;l ofLysis Solution to the center of the filter and allow it to soak whileperforming the next two steps (at least 5 min).</li>
	<li>Add 3 &#181;l of LCM Additive solution (provided in the kit) to the lysate and mix by vortexing for 5 sec. Centrifuge for 30 sec to collect the fluid at the bottom of the tube.</li>
	<li>Centrifuge the pre-wetted filter for ~30 sec at top speed to remove liquid.</li>
	<li>Add 1.25 volumes (in this case 129 &#181;l) of 100 % ethanol to the lysate mixture, gently vortex or pipette up and down. **This method will recover both large and small RNA species.</li>
	<li>Load the sample onto the pre-wetted micro filter, and centrifuge at 10,000 x g for 1 min to bind the RNA to the filter. Then wash micro filter with 180&#160;&#181;l of &#8220;Wash solution 1,&#8221; centrifuge at 10,000 x g for 1 min. 
	Note: From this point on all centrifugations should be done at 13,000 x g, or maximum speed.</li>
	<li>Wash filter twice with 180&#160;&#181;l of &#8221;Wash solutions 2 and 3&#8221;, respectively as directed by the kit protocol. Centrifuge for 30 sec, then discard the flow through, and spin the filter for 1 min to remove residual fluid and to dry the filter.</li>
	<li>Transfer filter to a new collection tube.</li>
	<li>Warm elution solution to -95 &#176;C (provided with the kit).</li>
	<li>Apply 10 &#181;l of preheated elution solution to the center of the filter, close the cap and store for 5 min at RT. Then centrifuge for 1 min to elute the RNA, and repeat this step one time to yield ~20 &#181;l of RNA.</li>
	<li>Perform the DNase I treatment for 15 min at 37 &#176;C.</li>
	<li>Quantify the RNA by spectrophotometer with absorbance at 260 nm. The ratio of optical density at wavelengths of 260 nm and 280 nm is used to assess the purity of the RNA (see Table 1)15.</li>
</ol>
6. Gene Expression Analysis
Note: Gene expression of long RNA species (like mRNAs and lncRNAs) is shown in Step 6.1 and short RNA species (like microRNAs) is shown in step 6.2. Different kits are available for RT-qPCR and the amount of RNA required varies by kit (as little as 10 ng). RNA sequencing analysis is described in 6.3.
<ol>
	<li>Reverse transcribe (RT) the RNA to cDNA mixing 5&#160;&#181;l of 20 ng/&#181;l RNA sample with 5 &#181;l of the RT master mix (RT buffer, dNTP&#8217;s, random primers, RT enzyme, RNase inhibitor). Incubate the reaction for 25 min at 25 &#176;C, then 120 min at 37 &#176;C and 5 min at 85 &#176;C.</li>
	<li>Dilute the RT reaction 1:10 with RNase free water and use 2.5 &#181;l per 10 &#181;l qPCR reaction with primers or primer/probe sets for the genes of interest. 
	Note: To facilitate analysis of partially degraded RNA It is advisable to use primers for shorter amplicons (less than 100 bp)16. (Table 2B)
	<ol>
		<li>For microRNA expression analysis run the RT reaction by mixing 5 &#181;l of 2-10 ng/&#181;l RNA sample with 5 &#181;l of the RT master mix. 
		Note: each commercially-available PCR-based microRNA detection technology requires the use of proprietary and specific RT primers.</li>
	</ol>
	</li>
	<li>Alternatively or in addition to RT-PCR, use next generation sequencing (RNA-seq or small-RNA-seq) to quantify different species of RNAs. Typically, use 500 ng of RNA for this procedure (Table 3) and carry out the method as indicated by the manufacturer and detection instrument.</li>
</ol>

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E., Haillot, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Patterns+of+spread+of+adenocarcinoma+in+the+prostate+as+related+to+cancer+volume.">Patterns of spread of adenocarcinoma in the prostate as related to cancer volume.</a> The Prostate. 49, 48-57 (2001).</li><li>Nonn, L., Vaishnav, A., Gallagher, L., Gann, P. 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. 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The authors have nothing to disclose.

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>
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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

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.

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

laser-capture-microdissection-human-prostatic-epithelium-for-rna

Research

JoVE Journal

Medicine

13.4K vistas.  University of Illinois at Chicago. 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.

Video: Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

Dissection of Human Vitreous Body Elements for Proteomic Analysis

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions between vitreous substructures and the retina underlie several vitreoretinal diseases, including retinal tear and detachment, macular pucker, macular hole, age-related macular degeneration, vitreomacular traction, proliferative vitreoretinopathy, proliferative diabetic retinopathy, and inherited vitreoretinopathies. 1,2 The molecular composition of the vitreous substructures is not known. Since the vitreous body is transparent with limited surgical access, it has been difficult to study its substructures at the molecular level. We developed a method to separate and preserve these tissues for proteomic and biochemical analysis. The dissection technique in this experimental video shows how to isolate vitreous base, anterior hyaloid, vitreous core, and vitreous cortex from postmortem human eyes. One-dimensional SDS-PAGE analyses of each vitreous component showed that our dissection technique resulted in four unique protein profiles corresponding to each substructure of the human vitreous body. Identification of differentially compartmentalized proteins will reveal candidate molecules underlying various vitreoretinal diseases.

This video shows an effective technique for differentiating and dissecting the various semi-transparent structures of the human vitreous body in post mortem eyes.

The vitreous is an optically clear, collagenous extracellular matrix that fills the inside of the eye and overlies the retina. 1,2 Abnormal interactions ...

Improved Protocol For Laser Microdissection Of Human Pancreatic Islets From Surgical Specimens

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected cells can be used for a variety of investigations, such as transcriptomic or proteomic studies, DNA assessment or chromosomal analysis 2,3. An especially challenging application of LMD is transcriptome analysis, which, due to the lability of RNA 4, can be particularly prominent when cells are dissected from tissues that are rich of RNases, such as the pancreas. A microdissection protocol that enables fast identification and collection of target cells is essential in this setting in order to shorten the tissue handling time and, consequently, to ensure RNA preservation. 
Here we describe a protocol for acquiring human pancreatic beta cells from surgical specimens to be used for transcriptomic studies 5. Small pieces of pancreas of about 0.5-1 cm3 were cut from the healthy appearing margins of resected pancreas specimens, embedded in Tissue-Tek O.C.T. Compound, immediately frozen in chilled 2-Methylbutane, and stored at -80 &deg;C until sectioning. Forty serial sections of 10 &mu;m thickness were cut on a cryostat under a -20 &deg;C setting, transferred individually to glass slides, dried inside the cryostat for 1-2 min, and stored at -80 &deg;C. 
Immediately before the laser microdissection procedure, sections were fixed in ice cold, freshly prepared 70% ethanol for 30 sec, washed by 5-6 dips in ice cold DEPC-treated water, and dehydrated by two one-minute incubations in ice cold 100% ethanol followed by xylene (which is used for tissue dehydration) for 4 min; tissue sections were then air-dried afterwards for 3-5 min. Importantly, all steps, except the incubation in xylene, were performed using ice-cold reagents - a modification over a previously described protocol 6. utilization of ice cold reagents resulted in a pronounced increase of the intrinsic autofluorescence of beta cells, and facilitated their recognition. For microdissection, four sections were dehydrated each time: two were placed into a foil-wrapped 50 ml tube, to protect the tissue from moisture and bleaching; the remaining two were immediately microdissected. This procedure was performed using a PALM MicroBeam instrument (Zeiss) employing the Auto Laser Pressure Catapulting (AutoLPC) mode. The completion of beta cell/islet dissection from four cryosections required no longer than 40-60 min. Cells were collected into one AdhesiveCap and lysed with 10 &mu;l lysis buffer. Each single RNA specimen for transcriptomic analysis was obtained by combining 10 cell microdissected samples, followed by RNA extraction using the Pico Pure RNA Isolation Kit (Arcturus). This protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their rapid and accurate recognition and collection. Further improvement of this procedure could enable the dissection of phenotypically different beta cells, with possible implications for better understanding the changes associated with type 2 diabetes.

Laser microdissection is a technique that allows the recovery of selected cells from minute amounts of parenchyma. Here we describe a protocol for acquiring human pancreatic islets from surgical specimens to be used for transcriptomic studies. Our protocol improves the intrinsic autofluorescence of human beta cells, thus facilitating their collection.

Laser microdissection (LMD) is a technique that allows the recovery of selected cells and tissues from minute amounts of parenchyma 1,2. The dissected ...

Formation of Human Prostate Epithelium Using Tissue Recombination of Rodent Urogenital Sinus Mesenchyme and Human Stem Cells

Progress in prostate cancer research is severely limited by the availability of human-derived and hormone-na&iuml;ve model systems, which limit our ability to understand genetic and molecular events underlying prostate disease initiation. Toward developing better model systems for studying human prostate carcinogenesis, we and others have taken advantage of the unique pro-prostatic inductive potential of embryonic rodent prostate stroma, termed urogenital sinus mesenchyme (UGSM). When recombined with certain pluripotent cell populations such as embryonic stem cells, UGSM induces the formation of normal human prostate epithelia in a testosterone-dependent manner. Such a human model system can be used to investigate and experimentally test the ability of candidate prostate cancer susceptibility genes at an accelerated pace compared to typical rodent transgenic studies. Since Human embryonic stem cells (hESCs) can be genetically modified in culture using inducible gene expression or siRNA knock-down vectors prior to tissue recombination, such a model facilitates testing the functional consequences of genes, or combinations of genes, which are thought to promote or prevent carcinogenesis.
The technique of isolating pure populations of UGSM cells, however, is challenging and learning often requires someone with previous expertise to personally teach. Moreover, inoculation of cell mixtures under the renal capsule of an immunocompromised host can be technically challenging. Here we outline and illustrate proper isolation of UGSM from rodent embryos and renal capsule implantation of tissue mixtures to form human prostate epithelium. Such an approach, at its current stage, requires in vivo xenografting of embryonic stem cells; future applications could potentially include in vitro gland formation or the use of induced pluripotent stem cell populations (iPSCs).

To unravel the earliest molecular mechanisms underlying prostate cancer initiation, novel and innovative human model systems and approaches are desperately needed. The potential of pre-prostatic urogenital sinus mesenchyme (UGSM) to induce pluripotent stem cell populations to form human prostate epithelium is a powerful experimental tool in prostate research.

Progress in prostate cancer research is severely  limited by the availability of human-derived and hormone-na&iuml;ve model systems,  which limit our ...

Renal Capsule Xenografting and Subcutaneous Pellet Implantation for the Evaluation of Prostate Carcinogenesis and Benign Prostatic Hyperplasia

New therapies for two common prostate diseases, prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend critically on experiments evaluating their hormonal regulation. Sex steroid hormones (notably androgens and estrogens) are important in PrCa and BPH; we probe their respective roles in inducing prostate growth and carcinogenesis in mice with experiments using compressed hormone pellets. Hormone and/or drug pellets are easily manufactured with a pellet press, and surgically implanted into the subcutaneous tissue of the male mouse host. We also describe a protocol for the evaluation of hormonal carcinogenesis by combining subcutaneous hormone pellet implantation with xenografting of prostate cell recombinants under the renal capsule of immunocompromised mice. Moreover, subcutaneous hormone pellet implantation, in combination with renal capsule xenografting of BPH tissue, is useful to better understand hormonal regulation of benign prostate growth, and to test new therapies targeting sex steroid hormone pathways.

We describe the manufacture of compressed hormone pellets, as well as subcutaneous surgical implantation into mice. This strategy can be combined with the growth of cell and tissue xenografts under the renal capsule of athymic nude mice to evaluate hormonal carcinogenesis and regulation of benign prostate growth.

New therapies for two common prostate diseases,  prostate cancer (PrCa) and benign prostatic hyperplasia (BPH), depend  critically on experiments ...

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tg(fli:EGFP) Zebrafish Retinal Vasculature

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of diabetic microvascular complications one of the key research fields of the next decades. Specialized, targeted therapy and novel therapeutic drugs are needed to manage the increasing number of patients at risk of vision-loss. The zebrafish is an established animal model for developmental research questions with increasing relevance for modeling metabolic multifactorial disease processes. The advantages of the species allow for optimal visualization and high throughput drug screening approaches, combined with the strong ability to knock out genes of interest. Here, we describe a protocol which will allow easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis or vessel damage. This is achieved via dissection of the zebrafish retina and whole-mounting of the tissue. Visualization of the exposed vessels is then achieved via confocal microscopy of the green EGFP reporter expressed in the adult retinal vasculature. Correct handling of the tissue will lead to better outcomes and less internal vessel breakage to assure the visualization of the unaltered vascular structure. The method can be utilized in zebrafish models of retinal vasculopathy linked to changes in the vessel architecture as well as neoangiogenesis.

Studying Diabetes Through the Eyes of a Fish: Microdissection, Visualization, and Analysis of the Adult tgfli:EGFP Zebrafish Retinal Vasculature

Here, we discuss a method protocol which will allow an easy analysis of the adult tg(fli:EGFP) zebrafish retinal vasculature as a fast read-out in settings of long-term vascular pathologies linked to neoangiogenesis and structural changes.

Diabetic retinopathy is the leading cause of blindness among middle-aged adults. The rising prevalence of diabetes worldwide will make the prevention of ...

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate (HoLEP).

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the quality of life. BPH includes 2 major treatments: medication and surgery. Surgical treatment is often the most effective and final intervention. Holmium laser enucleation of the prostate (HoLEP), one of the most efficient surgical procedures for BPH, is conducted transurethrally. During surgery, the most important and most difficult part is to locate the surgical capsule of the prostate, which is consistent to the idea of anatomic surgery. Different skills may be needed in treating different parts of the prostate. HoLEP tends to be efficient and safe with good hemostatic properties, and able to treat bladder calculus, which may be the complications of BPH. The technique is particularly suitable for prostate of varying volumes and sizes. Indeed, HoLEP also presents certain disadvantages, such as a long learning curve and costly equipment. Regardless, this method may be the "new standard" and the excellent method for the surgical treatment of BPH.

Surgical Treatment for Benign Prostatic Hyperplasia: Holmium Laser Enucleation of the Prostate HoLEP.

Here we present a safe and efficient protocol, holmium laser enucleation of the prostate, to treat benign prostatic hyperplasia.

Benign prostatic hyperplasia (BPH) commonly occurs among elderly males. The condition causes symptoms in the lower urinary tract, thereby reducing the ...

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry (LCM-LC/MS)

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel therapeutics are urgently required. The ability of dosed TB drugs to reach and sterilize bacteria within poorly-vascularized necrotic regions (caseum) of pulmonary granulomas is crucial for successful therapeutic intervention. Effective therapeutic regimens must therefore contain drugs with favorable caseum penetration properties. Current LC/MS methods for quantifying drug levels in biological tissues have limited spatial resolution capabilities, making it difficult to accurately determine absolute drug concentrations within small tissue compartments such as those found within necrotic granulomas. Here we present a protocol combining laser capture microdissection (LCM) of pathologically-distinct tissue regions with LC/MS quantification. This technique provides absolute quantification of drugs within granuloma caseum, surrounding cellular lesion and uninvolved lung tissue and, therefore, accurately determines whether bactericidal concentrations are being achieved. In addition to tuberculosis research, the technique has many potential applications for spatially-resolved quantification of drugs in diseased tissues.

Spatial Quantification of Drugs in Pulmonary Tuberculosis Lesions by Laser Capture Microdissection Liquid Chromatography Mass Spectrometry LCM-LC/MS

Here, we describe a protocol using laser capture microdissection coupled with LC/MS analysis to spatially-quantify drug distributions within pulmonary tuberculosis granulomas. The approach has broad applicability to quantifying drug concentrations within tissues at high spatial detail.

Tuberculosis is still a leading cause of morbidity and mortality worldwide. Improvements to existing drug regimens and the development of novel ...

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a promising therapeutic target in many disorders, such as autoimmune diseases, atherosclerosis, and cancer. RNAi-mediated gene silencing is a valuable approach of choice to probe and manipulate macrophage function; however, the transfection of macrophages with siRNA is often considered to be technically challenging, and, at present, few methodologies dedicated to the siRNA transfer to macrophages are available. Here, we present a protocol of using polyethyleneimine-coated superparamagnetic iron oxide nanoparticles (PEI-SPIONs) as a vehicle for the targeted delivery of siRNA to macrophages. PEI-SPIONs are capable of binding and completely condensing siRNA when the Fe:siRNA weight ratio reaches 4 and above. In vitro, these nanoparticles can efficiently deliver siRNA into primary macrophages, as well as into the macrophage-like RAW 264.7 cell line, without compromising cell viability at the optimal dose for transfection, and, ultimately, they induce siRNA-mediated target gene silencing. Apart from being used for in vitro siRNA transfection, PEI-SPIONs are also a promising tool for delivering siRNA to macrophages in vivo. In view of its combined features of magnetic property and gene-silencing ability, systemically administered PEI-SPION/siRNA particles are expected not only to modulate macrophage function but also to enable macrophages to be imaged and tracked. In essence, PEI-SPIONs represent a simple, safe, and effective nonviral platform for siRNA delivery to macrophages both in vitro and in vivo.

Polyethyleneimine-coated Iron Oxide Nanoparticles as a Vehicle for the Delivery of Small Interfering RNA to Macrophages In Vitro and In Vivo

We describe a method of using polyethyleneimine (PEI)-coated superparamagnetic iron oxide nanoparticles for transfecting macrophages with siRNA. These nanoparticles can efficiently deliver siRNA to macrophages in vitro and in vivo and silence target gene expression.

Because of their critical role in regulating immune responses, macrophages have continuously been the subject of intensive research and represent a ...

Isolation of Viable Adipocytes and Stromal Vascular Fraction from Human Visceral Adipose Tissue Suitable for RNA Analysis and Macrophage Phenotyping

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release different bioactive molecules that control metabolic, hormonal, and immune processes; currently, it is unclear how these processes are regulated within the adipose tissue. Therefore, the development of methods evaluating the contribution of each cell population to the pathophysiology of adipose tissue is crucial. This protocol describes the isolation steps and provides the necessary troubleshooting guidelines for efficient isolation of viable mature adipocytes and SVF from human VAT biopsies in a single process, using a collagenase enzymatic digestion technique. Moreover, the protocol is also optimized to identify macrophage subsets and perform mature adipocyte RNA isolation for gene expression studies, which allows performing studies dissecting the interaction between these cell populations. Briefly, VAT biopsies are washed, minced mechanically, and digested to generate a single-cell suspension. After centrifugation, mature adipocytes are isolated by flotation from the SVF pellet. The RNA extraction protocol ensures a high yield of total RNA (including miRNAs) from adipocytes for downstream expression assays. Simultaneously, SVF cells are used to characterize macrophage subsets (pro- and anti-inflammatory phenotype) through flow cytometry analysis.

This protocol provides an efficient collagenase digestion method for isolation of viable adipocytes and stromal vascular fraction-SVF cells from human visceral fat in a single process, including methodology to obtain high-quality RNA from adipocytes and phenotypification of SVF-macrophages through staining of multiple membrane-bound markers for analysis by flow cytometry.

Visceral adipose tissue (VAT) is an active metabolic organ composed mainly of mature adipocytes and stromal vascular fraction (SVF) cells, which release ...

Retinal Pigment Epithelium Transplantation in a Non-human Primate Model for Degenerative Retinal Diseases

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These conditions include retinitis pigmentosa (RP) and advanced forms of age-related macular degeneration (AMD), such as geographic atrophy (GA). Together, these disorders represent a significant proportion of currently untreatable blindness globally. These unmet medical needs have generated heightened academic interest in developing methods of RPE replacement. Among the animal models commonly utilized for preclinical testing of therapeutics, the non-human primate (NHP) is the only animal model that has a macula. As it shares this anatomical similarity with the human eye, the NHP eye is an important and appropriate preclinical animal model for the development of advanced therapy medicinal products (ATMPs) such as RPE cell therapy.
This manuscript describes a method for the submacular transplantation of an RPE monolayer, cultured on a polyethylene terephthalate (PET) cell carrier, underneath the macula onto a surgically created RPE wound in immunosuppressed NHPs. The fovea-the central avascular portion of the macula-is the site of the greatest mechanical weakness during the transplantation. Foveal trauma will occur if the initial subretinal fluid injection generates an excessive force on the retina. Hence, slow injection under perfluorocarbon liquid (PFCL) vitreous tamponade is recommended with a dual-bore subretinal injection cannula at low intraocular pressure (IOP) settings to create a retinal bleb. 
Pretreatment with an intravitreal plasminogen injection to release parafoveal RPE-photoreceptor adhesions is also advised. These combined strategies can reduce the likelihood of foveal tears when compared to conventional techniques. The NHP is a key animal model in the preclinical phase of RPE cell therapy development. This protocol addresses the technical challenges associated with the delivery of RPE cellular therapy in the NHP eye.

The non-human primate (NHP) is an ideal model for studying human retinal cellular therapeutics due to the anatomical and genetic similarities. This manuscript describes a method for submacular transplantation of retinal pigment epithelial cells in the NHP eye and strategies to prevent intraoperative complications associated with macular manipulation.

Retinal pigment epithelial (RPE) transplantation holds great promise for the treatment of inherited and acquired retinal degenerative diseases. These ...