Name: Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis (Video) | JoVE Journal
Uploaded: 2015-11-26T15:00:00.000+00:00
Duration: 7 min 42 s
Description: Learn laser-capture microdissection to isolate pure prostatic epithelial cells from frozen prostate tissue for RNA analysis. This method enables precise gene expression studies in prostate cancer research.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

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

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...

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 ...

The overall goal of this laser capture micro dissection protocol is to isolate total RNA from pure populations of prostatic epithelial cell types from heterogeneous frozen prostate tissues for downstream gene expression analyses. This method can help analyze patient specimens and answer key questions in the prostate cancer research field, such as how RNA expression differs in benign and disease epithelial prosthetic tissue samples, or in clinical trial specimens. The main advantage of this technique is that laser capture micro dissection enables RNA isolation of homogeneous populations of epithelial or str cells from frozen prostate tissue.Generally, individuals new to this method will struggle to acquire high quality RNA because they do not know which steps are critical to maintain RNAs. Free conditions Begin by gathering all reagents and tools needed for the procedure. After allowing the frozen prostate tissue to equilibrate inside the cryostat for 30 minutes, squirt mounting medium into the bottom of the cassette and quickly mount the tissue using chilled forceps.Place the cassette into the chiller of the cryostat for five minutes to set the mounting medium. Once set, squirt frozen, mounting medium onto the chuck and carefully press the mounted tissue into it. Bottom side up.Allow the preparation to set for five minutes. Before placing the chuck into the holder, lock the handle into position and insert a new blade onto the cryostat. After unlocking the handle, carefully advance the sample until the desired tissue exposure is reached.When ready, adjust the cryostat to five microns and cut one or two sections. Place these sections onto a charged glass slide for hematin and eoin staining. Immediately place the slide into cold, 100%ethanol for two minutes.After this time, remove the slide and let it dry at room temperature. The h and d stained slide provides better visualization of nuclear textures and should be used by the pathologist to mark the areas of interest. Next, place a room temperature pen frame.Slide into a frame supporter, adjust the cryostat to 10 microns and create sections. One to four sections can be placed onto each slide, depending on the tissue size. Immediately plunge the slide into cold 100%ethanol.Remove the slide after two minutes and store in the freezer for later analysis. On the same day of the laser capture microdissection, retrieve the pen frame slides from the freezer working in an RNA free area. Hydrate the slides by gently dipping them into 90%ethanol for two minutes, then 75%ethanol for two minutes.Once hydrated, stain the prostate tissue by gently dipping the slides into 0.5%toine blue for between five to 30 seconds. Detain the tissue by washing the slides twice in depthy water for 15 seconds, followed by 75%ethanol for 30 seconds to three minutes. Lastly, transfer the pen frame slides to an RNA free dry container on ice.Before beginning the procedure, place a slide to dry on a slide warmer for 15 minutes. Dry only the slide that will be processed and store the rest on ice until needed. Turn on the LCM components in the following order.Microscope power, the laser key, the laser switch, and then the computer. Next, launch the laser microdissection software using the software. Prompt the microscope to load the slides and collection tubes.Click on unload sample holder and load the slide onto the slide holder with the tissue facing down. Insert the slide holder into the appropriate slot in the microscope and click continue. Next, click on unload collection tubes.Label and load tubes onto the tube holder and insert into the appropriate slot. Secure the caps and add 35 microliters of lysis buffer. Taking care to avoid bubbles.Once the collection caps are loaded, click okay. Use the software to select the position of the slide in the holder, and then select the collection cap to collect the specimen. Visualize and focus the slide through the computer at 10 x magnification.Use the software to calibrate the laser by selecting laser. Then calibrate, adjust the power aperture and speed of the laser by selecting laser, and then control. Continue to make adjustments until the laser cuts completely through the tissue.It is crucial to outline the areas of interest typically done by a board certified pathologist in order to accurately dissect the desire areas of the tissue subjected to LCM Using the eight by 11 h and e pathologist markup as a visual guide. Use the draw shape tool in the software to circumscribe the desired areas of epithelium in the view and avoid collecting any stroma. Click cut to start the laser.If an area is not completely cut, use the move and cut tool to go over it manually. Continue moving the objective to new views and repeat laser cutting until the slide is completely dissected or the desired amount of tissue has been collected. When complete, carefully remove the tubes from the collection holder and close the caps being mindful of the lysis, buffer and tissue in the caps.Briefly, centrifuge for 30 seconds to collect the liquid and tissue in the bottom of the tube. Incubate the samples at 42 degrees Celsius for 30 minutes and store at minus 80 degrees Celsius until ready for RNA isolation. These examples show representative toluidine blue stained prostate sections on a pen frame slide before and after laser capturing benign epithelium cell type specific controls.Confirm the specificity of the laser captured area of the tissue. Benign epithelium and stroma. Do not express the prostate cancer specific gene, A-M-A-C-R.The stroma did not express the epithelial specific gene and KX 3.1 While attempting this procedure. It's important to remember to work in an RNA free environment to prevent RNA degradation. After watching this video, you should have a good understanding of how to isolate RNA from homogeneous populations of epithelial or storm cells from frozen tissue for numerous downstream analysis techniques.Don't forget that working with a cryostat can be extremely hazardous in precautions, such as good training and extremely good attention to the blade should always be taken while performing this procedure.

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

Research

JoVE Journal

Medicine

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.

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

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.

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

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.

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

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).

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

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

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.

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

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.

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

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 of Mouse Embryonic Cartilage and Bone for Gene Expression Analysis

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.

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

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.

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

This protocol describes a method to isolate RNA from defined histological regions of paraformaldehyde-fixed, Optimal Cutting Temperature compound-embedded mouse liver tissue using laser capture microdissection, enabling targeted downstream gene expression analysis.

Laser Capture Microdissection of Paraformaldehyde-Fixed Mouse Liver Tissue for RNA Analysis

Learn laser-capture microdissection to isolate pure prostatic epithelial cells from frozen prostate tissue for RNA analysis. This method enables precise gene expression studies in prostate cancer research.

Laser-capture Microdissection of Human Prostatic Epithelium for RNA Analysis

Explore More Videos