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>

<ol><li>Schauer, I. G., Rowley, D. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+functional+role+of+reactive+stroma+in+benign+prostatic+hyperplasia%5BTitle%5D)+AND+%22Differentiation%22%5BJournal%5D)">The functional role of reactive stroma in benign prostatic hyperplasia.</a> Differentiation. 82, 200-210 (2011).</li>
<li>Peehl, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Primary+cell+cultures+as+models+of+prostate+cancer+development%5BTitle%5D)+AND+%22Endocrine-related+cancer%22%5BJournal%5D)">Primary cell cultures as models of prostate cancer development.</a> Endocrine-related cancer. 12, 19-47 (2005).</li>
<li>McNeal, J. E., Haillot, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Patterns+of+spread+of+adenocarcinoma+in+the+prostate+as+related+to+cancer+volume%5BTitle%5D)+AND+%22The+Prostate%22%5BJournal%5D)">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/pubmed?term=((mRNA+and+micro-RNA+expression+analysis+in+laser-capture+microdissected+prostate+biopsies%3A+valuable+tool+for+risk+assessment+and+prevention+trials%5BTitle%5D)+AND+%22Experimental+and+molecular+pathology%22%5BJournal%5D)">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/pubmed?term=((Global+transcriptome+analysis+of+formalin-fixed+prostate+cancer+specimens+identifies+biomarkers+of+disease+recurrence%5BTitle%5D)+AND+%22Cancer+research%22%5BJournal%5D)">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/pubmed?term=((Protein-coding+and+microRNA+biomarkers+of+recurrence+of+prostate+cancer+following+radical+prostatectomy%5BTitle%5D)+AND+%22The+American+journal+of+pathology%22%5BJournal%5D)">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/pubmed?term=((Differential+expression+and+regulation+of+vitamin+D+hydroxylases+and+inflammatory+genes+in+prostate+stroma+and+epithelium+by+1%2C25-dihydroxyvitamin+D+in+men+with+prostate+cancer+and+an+in+vitro.+model%5BTitle%5D)+AND+%22The+Journal+of+steroid+biochemistry+and+molecular+biology%22%5BJournal%5D)">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/pubmed?term=((Tumor+suppressor+microRNAs%2C+miR-100+and+-125b%2C+are+regulated+by+1%2C25-dihydroxyvitamin+D+in+primary+prostate+cells+and+in+patient+tissue%5BTitle%5D)+AND+%22Cancer+prevention+research%22%5BJournal%5D)">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/pubmed?term=((miR-183-96-182+cluster+is+overexpressed+in+prostate+tissue+and+regulates+zinc+homeostasis+in+prostate+cells%5BTitle%5D)+AND+%22The+Journal+of+biological+chemistry%22%5BJournal%5D)">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/pubmed?term=((Maintaining+RNA+integrity+in+a+homogeneous+population+of+mammary+epithelial+cells+isolated+by+Laser+Capture+Microdissection%5BTitle%5D)+AND+%22BMC+cell+biology%22%5BJournal%5D)">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/pubmed?term=((Analysis+of+gene+expression+in+prostate+cancer+epithelial+and+interstitial+stromal+cells+using+laser+capture+microdissection%5BTitle%5D)+AND+%22BMC+cancer%22%5BJournal%5D)">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/pubmed?term=((Comparative+microRNA+profiling+of+prostate+carcinomas+with+increasing+tumor+stage+by+deep+sequencing%5BTitle%5D)+AND+%22Molecular+cancer+research+%3A+MCR%22%5BJournal%5D)">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/pubmed?term=((Endogenous+siRNAs+and+noncoding+RNA-derived+small+RNAs+are+expressed+in+adult+mouse+hippocampus+and+are+up-regulated+in+olfactory+discrimination+training%5BTitle%5D)+AND+%22Rna%22%5BJournal%5D)">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/pubmed?term=((Expression+profile+analysis+of+microRNAs+in+prostate+cancer+by+next-generation+sequencing%5BTitle%5D)+AND+%22The+Prostate%22%5BJournal%5D)">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/pubmed?term=((Comparison+of+six+RNA+extraction+methods+for+the+detection+of+classical+swine+fever+virus+by+real-time+and+conventional+reverse+transcription-PCR%5BTitle%5D)+AND+%22Journal+of+veterinary+diagnostic+investigation+%3A+official+publication+of+the+American+Association+of+Veterinary+Laboratory+Diagnosticians%2C+Inc%22%5BJournal%5D)">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/pubmed?term=((Quantitative+assessment+of+short+amplicons+in+FFPE-derived+long-chain+RNA%5BTitle%5D)+AND+%22Scientific+reports%22%5BJournal%5D)">Quantitative assessment of short amplicons in FFPE-derived long-chain RNA.</a> Scientific reports. 4, 7246(2014).</li>
</ol>

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&amp;nbsp;</td><td>MBP</td><td>7005-11</td><td></td></tr><tr><td>PEN-membrane 4.0 mm slides&amp;nbsp;</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&amp;reg; 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&amp;reg;-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&amp;nbsp;</td><td>Ricca Chemical Company</td><td>3536-16</td><td></td></tr><tr><td>Eosin-Y</td><td>Richard Allan Scientific&amp;nbsp;</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

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 ...

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Research

JoVE Journal

Medicine

13.4K Views.  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

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

Sequencing Small Non-coding RNA from Formalin-fixed Tissues and Serum-derived Exosomes from Castration-resistant Prostate Cancer Patients

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results in cancer regression. However, in a significant number of cases, the disease progresses to advanced, castration-resistant prostate cancer (CRPC), which has limited therapeutic options and is often aggressive. Distant metastasis is mostly observed at this stage of the aggressive disease. CRPC is treated by a second generation of AR pathway inhibitors that improve survival initially, followed by the emergence of therapy resistance. Neuroendocrine prostate cancer (NEPC) is a rare variant of prostate cancer (PCa) that often develops as a result of therapy resistance via a transdifferentiation process known as neuroendocrine differentiation (NED), wherein PCa cells undergo a lineage switch from adenocarcinomas and show increased expression of neuroendocrine (NE) lineage markers. In addition to the genomic alterations that drive the progression and transdifferentiation to NEPC, epigenetic factors and microenvironmental cues are considered essential players in driving disease progression. This manuscript provides a detailed protocol to identify the epigenetic drivers (i.e., small non-coding RNAs) that are associated with advanced PCa. Using purified microRNAs from formalin-fixed paraffin-embedded (FFPE) metastatic tissues and corresponding serum-derived extracellular vesicles (EVs), the protocol describes how to prepare libraries with appropriate quality control for sequencing microRNAs from these sample sources. Isolating RNA from both FFPE and EVs is often challenging because most of it is either degraded or is limited in quantity. This protocol will elaborate on different methods to optimize the RNA inputs and cDNA libraries to yield most specific reads and high-quality data upon sequencing.

Therapy resistance often develops in patients with advanced prostate cancer, and in some cases, cancer progresses to a lethal subtype called neuroendocrine prostate cancer. Assessing the small non-coding RNA-mediated molecular changes that facilitate this transition would allow better disease stratification and identification of causal mechanisms that lead to development of neuroendocrine prostate cancer.

Ablation of androgen receptor (AR) signaling by androgen deprivation is the goal of the first line of therapy for prostate cancer that initially results ...

Cancer Research

Microarray-based Identification of Individual HERV Loci Expression: Application to Biomarker Discovery in Prostate Cancer

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, particularly in low dosage values1&#8203;&#8203;. &#8216;How to use PSA&#39; remains a current issue, either for diagnosis as a gray zone corresponding to a concentration in serum of 2.5-10 ng/ml which does not allow a clear differentiation to be made between cancer and noncancer2 or for patient follow-up as analysis of post-operative PSA kinetic parameters can pose considerable challenges for their practical application3,4. Alternatively, noncoding RNAs (ncRNAs) are emerging as key molecules in human cancer, with the potential to serve as novel markers of disease, e.g. PCA3 in prostate cancer5,6 and to reveal uncharacterized aspects of tumor biology. Moreover, data from the ENCODE project published in 2012 showed that different RNA types cover about 62% of the genome. It also appears that the amount of transcriptional regulatory motifs is at least 4.5x higher than the one corresponding to protein-coding exons. Thus, long terminal repeats (LTRs) of human endogenous retroviruses (HERVs) constitute a wide range of putative/candidate transcriptional regulatory sequences, as it is their primary function in infectious retroviruses. HERVs, which are spread throughout the human genome, originate from ancestral and independent infections within the germ line, followed by copy-paste propagation processes and leading to multicopy families occupying 8% of the human genome (note that exons span 2% of our genome). Some HERV loci still express proteins that have been associated with several pathologies including cancer7-10. We have designed a high-density microarray, in Affymetrix format, aiming to optimally characterize individual HERV loci expression, in order to better understand whether they can be active, if they drive ncRNA transcription or modulate coding gene expression. This tool has been applied in the prostate cancer field (Figure 1).

Human endogenous retroviruses (HERV), which occupy 8% of the human genome, retain scarce coding capacities but a hundred thousand long terminal repeats (LTRs). A custom Affymetrix microarray was designed to identify individual HERV locus expression and was used on prostate cancer tissues as a proof of concept for future clinical studies.

The prostate-specific antigen (PSA) is the main diagnostic biomarker for prostate cancer in clinical use, but it lacks specificity and sensitivity, ...

Laser Capture Microdissection of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and molecular complexity of skeletal elements increases with development. Tissue heterogeneity, such as at the interface of cartilaginous and osseous elements with each other or with surrounding tissues, is one obstacle to the study of developing cartilage and bone. Our protocol provides a rapid method of tissue processing and isolation of cartilage and bone that yields high quality RNA for gene expression analysis. Fresh frozen tissues of mouse embryos are sectioned and brief cresyl violet staining is used to visualize cartilage and bone with colors distinct from surrounding tissues. Slides are then rapidly dehydrated, and cartilage and bone are isolated subsequently by LCM. The minimization of exposure to aqueous solutions during this process maintains RNA integrity. Mouse Meckel&rsquo;s cartilage and mandibular bone at E16.5 were successfully collected and gene expression analysis showed differential expression of marker genes for osteoblasts, osteocytes, osteoclasts, and chondrocytes. High quality RNA was also isolated from a range of tissues and embryonic ages. This protocol details sample preparation for LCM including cryoembedding, sectioning, staining and dehydrating fresh frozen tissues, and precise isolation of cartilage and bone by LCM resulting in high quality RNA for transcriptomic analysis.

This protocol describes laser capture microdissection for the isolation of cartilage and bone from fresh frozen sections of the mouse embryo. Cartilage and bone can be rapidly visualized by cresyl violet staining and collected precisely to yield high quality RNA for transcriptomic analysis.

Laser capture microdissection (LCM) is a powerful tool to isolate specific cell types or regions of interest from heterogeneous tissues. The cellular and ...

Developmental Biology

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

Optimization of Laser-Capture Microdissection for the Isolation of Enteric Ganglia from Fresh-Frozen Human Tissue

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). We have identified five steps in the workflow that are crucial for obtaining RNA isolates from enteric ganglia with sufficiently high quality and quantity for RNA-seq. First, when preparing intestinal tissue, each sample must have all excess liquid removed by blotting prior to flattening the serosa as much as possible across the bottom of large base molds. Samples are then quickly frozen atop a slurry of dry ice and 2-methylbutane. Second, when sectioning the tissue, it is important to position cryomolds so that intestinal sections parallel the full plane of the myenteric plexus, thereby yielding the greatest surface area of enteric ganglia per slide. Third, during LCM, polyethylene napthalate (PEN)-membrane slides offer the greatest speed and flexibility in outlining the non-uniform shapes of enteric ganglia when collecting enteric ganglia. Fourth, for distinct visualization of enteric ganglia within sections, ethanol-compatible dyes, like Cresyl Violet, offer excellent preservation of RNA integrity relative to aqueous dyes. Finally, for the extraction of RNA from captured ganglia, we observed differences between commercial RNA extraction kits that yielded superior RNA quantity and quality, while eliminating DNA contamination. Optimization of these factors in the current protocol greatly accelerates the workflow and yields enteric ganglia samples with exceptional RNA quality and quantity.

The goal of this protocol is to obtain high-integrity RNA samples from enteric ganglia isolated from unfixed, freshly-resected human intestinal tissue using laser capture microdissection (LCM). This protocol involves preparing flash-frozen samples of human intestinal tissue, cryosectioning, ethanolic staining and dehydration, LCM, and RNA extraction.

The purpose of this method is to obtain high-integrity RNA samples from enteric ganglia collected from unfixed, freshly-resected human intestinal tissue ...

Neuroscience

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

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires precise identification of cells subpopulations from a heterogeneous tissue. Identification of cells of interest for LCM is usually based on morphological criteria or on fluorescent protein reporters. The combination of LCM and rapid immunolabeling offers an alternative and efficient means to visualize specific cell types and to isolate them from surrounding tissue. High-quality RNA can then be extracted from a pure cell population and further processed for downstream applications, including RNA-sequencing, microarray or qRT-PCR. This approach has been previously performed and briefly described in few publications. The goal of this article is to illustrate how to perform rapid immunolabeling of a cell population while keeping RNA integrity, and how to isolate these specific cells using LCM. Herein, we illustrated this multi-step procedure by immunolabeling and capturing dopaminergic cells in brain tissue from one-day-old mice. We highlight key critical steps that deserve special consideration. This protocol can be adapted to a variety of tissues and cells of interest. Researchers from different fields will likely benefit from the demonstration of this approach.

Gene expression analysis of a subset of cells in a specific tissue represents a major challenge. This article describes how to isolate high-quality total RNA from a specific cell population by combining a rapid immunolabeling method with laser capture microdissection.

Laser capture microdissection (LCM) allows the isolation of specific cells from thin tissue sections with high spatial resolution. Effective LCM requires ...

Biology

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

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

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

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

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

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

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

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

Application of Laser Microdissection to Uncover Regional Transcriptomics in Human Kidney Tissue

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and depth of this technology and extending it to the level of cells within the tissue is needed. Although the use of single nuclear and single cell RNA sequencing has become widespread, the expression signatures of cells obtained from tissue dissociation do not maintain spatial context. Laser microdissection (LMD) based on specific fluorescent markers would allow the isolation of specific structures and cell groups of interest with known localization, thereby enabling the acquisition of spatially-anchored transcriptomic signatures in kidney tissue. We have optimized an LMD methodology, guided by a rapid fluorescence-based stain, to isolate five distinct compartments within the human kidney and conduct subsequent RNA sequencing from valuable human kidney tissue specimens. We also present quality control parameters to enable the assessment of adequacy of the collected specimens. The workflow outlined in this manuscript shows the feasibility of this approach to isolate sub-segmental transcriptomic signatures with high confidence. The methodological approach presented here may also be applied to other tissue types with substitution of relevant antibody markers.

We describe a protocol for laser microdissection of sub-segments of the human kidney, including the glomerulus, proximal tubule, thick ascending limb, collecting duct and interstitium. The RNA is then isolated from the obtained compartments and RNA sequencing is carried out to determine changes in the transcriptomic signature within each sub-segment.

Gene expression analysis of human kidney tissue is an important tool to understand homeostasis and disease pathophysiology. Increasing the resolution and ...

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

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