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).

Tutti i tessuti umani usati per questi esperimenti sono stati acquisiti tramite un protocollo Institutional Review Board approvato e / o di esenzione presso University of Illinois a Chicago. 1. Sezione freschi congelati prostata su PEN-scivolo e sulla Charged vetrino <ol><li> Il giorno prima o qualche ora prima sezionare il campione, attrezzi puliti (ad esempio, spazzola, pinze, coplins, lame, scivoli PEN-incorniciati (se non è già RNasi gratuito), un ETOH marcatore di sicurezza e una matita) e l&#39;interno di una RT criostato con la soluzione RNase-decontaminazione utilizzando uno spray e di laboratorio-salviette. <ol><li> Lasciare la soluzione-RNasi decontaminazione a sedere per 5 minuti prima asciugandosi, sciacquare con depcH 2 0 da rimuovere sinistra su una soluzione e un risciacquo finale con il 70% EtOH (preparato con depcH 2 0) RNasi-decontaminazione. Nota: E &#39;essenziale mantenere uno spazio di lavoro RNasi-free. Tutti coplins, strumenti, mezzo di montaggio congelato, lame e diapositive usati devono essere utilizzati solo per RNasi lavoro gratise mai usato in un ambiente privo di serie non RNasi. Tutti gli strumenti devono essere trattati con una soluzione di decontaminazione RNasi prima di ogni esperimento. </li></ol></li><li> Raffreddare criostato a -24 ° C. </li><li> Posizionare i seguenti puliti strumenti gratuiti RNasi all&#39;interno criostato raffreddare almeno 30 minuti prima di sezionamento: slide Coplin-riempita con 100% di etanolo, gli strumenti, le piccole cassette di tessuto congelato e mandrino montaggio. </li><li> Recupera tessuto prostatico congelato da crioconservazione e posto in una schiuma secchio pieno di ghiaccio secco per il trasporto. Collocare il tessuto in criostato equilibrare al criostato temperatura per almeno 30 minuti. Nota: tessuti della prostata umane per laser-capture micro-dissezione possono essere sia fresco congelato o fissato in formalina e incluso in paraffina (FFPE). I vincoli di tempo e preparazione del vetrino sono leggermente diverse tra le fonti di tessuto. Qui si descrivono i passaggi per sezioni congelate. </li><li> Lavorare con un tessuto alla volta, e schizzare mezzo di montaggio congelato nel bottom della cassetta e rapidamente montare il tessuto prostatico congelato nel mezzo di montaggio congelati utilizzando le pinze refrigerati. </li><li> Posizionare la cassetta con il tessuto sul refrigeratore in criostato per 5 minuti per impostare montante congelato. </li><li> Bagnate una piccola quantità di mezzo di montaggio congelato sul mandrino e premere delicatamente il tessuto montato lato basso verso l&#39;alto sul montante congelato. Let impostato nel criostato per 5 minuti per consentire la soluzione di montaggio congelato solidificare completamente. </li><li> Posizionare mandrino nel supporto e stringere. </li><li> Bloccare la maniglia in posizione di blocco e posizionare una nuova lama sul criostato. Per gli endpoint molecolari downstream che comprendono una fase di amplificazione (cioè, qPCR) è consigliabile utilizzare una nuova lama per ogni paziente per prevenire la contaminazione (o spostare la lama verso utilizzare parte della lama per il campione successivo). </li><li> Sbloccare la maniglia e far avanzare con attenzione il tessuto a mano o con un pedale per uniformare ed esporre il tessuto con 50 μm sezioni fino all&#39;esposizione tessuto desiderato viene raggiunto. </li><li> Regolare criostato a 5 micron, tagliare una o due sezioni e metterli su vetrino addebitato per ematossilina eosina (H &amp; E) colorazione (vedi punto 2). </li><li> Porre immediatamente il vetrino al freddo 100% di etanolo per 2 min. </li><li> Rimuovere il vetrino caricato da etanolo e lasciare asciugare a temperatura ambiente prima di procedere alla colorazione H &amp; E (punto 2). </li><li> Posizionare RT PEN-frame di diapositive su un supporter telaio. </li><li> Regolare criostato a 10 micron e posizionare le sezioni sui vetrini telaio PEN RT. Da uno a quattro sezioni può essere posto su ciascun vetrino a seconda delle dimensioni del tessuto. </li><li> Immergere immediatamente il vetrino al freddo 100% di etanolo per 2 min. </li><li> Rimuovere i vetrini PEN-telaio da etanolo e conservare il vetrino in una scatola di diapositive in una scatola in un essiccatore -80 ° C freezer fino al momento LCM. Tre giorni è il tempo massimo di conservazione per il recupero ottimale RNA. </li><li> Procedere con la fase 2 per solo il vetro carica scivolòe. Procedere con il passaggio 3 per PEN-telai per diapositive. </li></ol> 2. ematossilina e eosina-Y (H &amp; E) Stain per istologico Mark-up Nota: Questo se per il tessuto sul vetrino carica e NON la slitta PEN-cornice per LCM. <ol><li> Idratare le sezioni sul vetrino carica immergendo il vetrino attraverso etanolo classificato come segue: immergerlo due volte in 100% di etanolo per 3 min, due volte in 95% di etanolo per 3 min, una volta in 70% di etanolo per 3 min e due volte in acqua distillata H 2 O per 3 min. <ol><li> Preparare la soluzione alcolica acida: 99 ml di etanolo al 70% + 1 ml 1% e 0,1% di HCl soluzione di bicarbonato di sodio: 0,1 g di bicarbonato di sodio in 100 ml di acqua distillata H 2 O </li></ol></li><li> Immergere il vetrino in ematossilina Gill II (0,4%) per 5 min. </li><li> Lavare eccesso macchia in costante rubinetto aperto H 2 O per 3 min. </li><li> Immergere il vetrino 10 volte in alcol acido </li><li> Lavare il vetrino in esecuzione rubinetto H 2 O per 3 min. </li><li> Immergere il vetrinonello 0,1% soluzione di bicarbonato di sodio per 30-60 secondi per accendere il colore violaceo a blu. </li><li> Lavare il vetrino in esecuzione rubinetto H 2 O per 3 min. </li><li> Sciacquare il vetrino in etanolo al 95% con 10 tuffi. </li><li> Immergere il vetrino in tempi eosina-Y 2-3 per 15 sec totale. </li><li> Disidratare il vetrino attraverso etanolo classificato come segue: immergere il vetrino due volte in etanolo al 95%, due volte in 100% di etanolo, e due volte a xilene (assicurarsi che il tessuto appare chiaro che indica la disidratazione completa). </li><li> Montare la slitta con non acquoso dura mezzo di montaggio e copertura di slittamento permanente. </li><li> Lasciare asciugare il vetrino per 30 min. </li><li> Diapositiva scansione con qualsiasi scanner intera diapositiva e stampare una grande, un&#39;immagine ad alta risoluzione su 8 &quot;x 11&quot;. </li><li> Outline aree di interesse (in genere fatto da un consiglio di anatomopatologo certificata) con un pennarello. Questa marcatura è la guida per la procedura di LCM. </li></ol> 3. Toluidine Blu colorazione dei vetrini PEN-telaio Nota: Thsi è fatto lo stesso giorno il LCM. Utilizzare depcH 2 O acqua per tutte le soluzioni. Completare tutti i passaggi zona di libero RNasi. Tutti coplins devono essere puliti con la soluzione RNase-decontaminazione. <ol><li> Il giorno della LCM rimuovere i vetrini PEN-frame da -80 ° C freezer e idratare da diapositive immersione delicatamente nel 90% EtOH per 2 minuti poi il 75% EtOH per 2 min. <ol><li> Preparare 0,5% toluidina blu sciogliendo in acqua per biologia molecolare, quindi filtrare sterilizzare attraverso un filtro 0,2 micron e conservare a temperatura ambiente. Questa soluzione può essere riutilizzato per un massimo di 6 mesi. Nota: E &#39;altamente consigliato di preparare la soluzione prima come la filtrazione può prendere un paio di ore. </li></ol></li><li> Colorare il tessuto prostatico mediante immersione delicatamente diapositive in 0,5% toluidina blu per 5-30 secondi. Decolorare il tessuto mediante lavaggio vetrini 2 volte in depcH 2 O per 15 secondi seguiti da 75% EtOH per 30 sec a 3 min. Nota: macchia e Decolorare tempi possono essere regolati per garantire una buona colorazione. Prostata tende a oltre macchia. </li><li&gt; trasferimento diapositive PEN-struttura di riparo RNAase-gratuito su ghiaccio. </li></ol> 4. laser-capture Micro-dissezione (LCM) <ol><li> Immediatamente prima LCM, scivolo secco su un vetrino più caldo a 42 ° C per 15 min. Secco solo la diapositiva che verrà elaborato e memorizzare il resto sul ghiaccio fino al momento per loro. </li><li> Accendere LCM e computer in questo ordine, di alimentazione del microscopio, la chiave laser, l&#39;interruttore laser e quindi il computer. Avviare il software Microdissezione laser. </li><li> Utilizzare il software per controllare il microscopio per caricare le diapositive e tubi di raccolta. Clicca su &quot;supporto del campione scaricare&quot;, caricare la slitta con le sezioni sul supporto vetrino da microscopio con il tessuto rivolto verso il basso e inserire il supporto diapositive nello slot appropriato al microscopio, quindi fare clic su &quot;continua&quot;. <ol><li> Clicca su &quot;tubi di raccolta scarico&quot;, etichetta e caricare 0,5 ml provette sul supporto tubo e inserirla nello slot appropriato nello strumento. Una voltatappi sono garantiti, aggiungere 35 ml di tampone Lysis per i tappi di raccolta e cercare di evitare le bolle. Quindi fare clic su &quot;OK&quot;. Nota: se l&#39;evaporazione è un problema, diluire il tampone come segue: tampone di lisi 25 microlitri con 10 microlitri depcH2O. </li></ol></li><li> Nel software, selezionare la posizione in cui la diapositiva è stata posta nel portavetrini. Selezionare il cappuccio di raccolta per raccogliere il campione LCM. </li><li> Visualizza e mettere a fuoco la slitta attraverso il computer a 10X. Utilizzare il software per calibrare il laser selezionando &quot;Laser&quot; e poi &quot;calibrare&quot;. Regolare la potenza, apertura e la velocità del laser, selezionando &quot;laser&quot; poi &quot;controllo&quot; per consentire il raggio laser per tagliare l&#39;area selezionata completo ed efficiente. Ripetere le regolazioni fino tagli laser completamente attraverso il tessuto. </li><li> Utilizzando il mark-up 8 &quot;x 11&quot; patologo come guida, utilizzare lo strumento &quot;disegnare forma&quot; circoscrivere le aree desiderate di epithelium nella vista ed evitare la raccolta di qualsiasi stroma. Nota: le aree multipli possono essere selezionati all&#39;interno di un punto di vista, ma non si spostano in un altro senza prima vista taglio facendo clic sullo strumento &quot;taglio forma&quot;. <ol><li> Se un&#39;area non viene completamente tagliato dal laser utilizzare lo strumento &quot;circolare e di taglio&quot; di andare oltre manualmente. Continua spostando l&#39;obiettivo di nuove viste e ripetere taglio laser fino slitta è completamente sezionato o la quantità desiderata di tessuto è stato raccolto (tipicamente 100 ghiandole benigne resa 150-500 ng di RNA). (vedi esempio in figura 1A). </li></ol></li><li> Rimuovere con attenzione i tubi dal titolare di raccolta e chiudere i tappi, memori del tampone di lisi e del tessuto nelle calotte. Brevemente centrifugare per 30 sec per raccogliere liquidi e tessuto del fondo della provetta. Incubare i campioni a 42 ° C per 30 min e conservare a -80 ° C fino al momento per l&#39;isolamento dell&#39;RNA. </li></ol> 5. totale isolamento di RNA con un FilKit base ter-e analisi di qualità <ol><li> Scongelare le provette su ghiaccio e portare il volume della soluzione a 100 microlitri. </li><li> Pre-bagnare il micro filtro di un kit di isolamento dell&#39;RNA applicando 30 microlitri ofLysis Soluzione al centro del filtro e lasciare in ammollo whileperforming le due fasi successive (almeno 5 minuti). </li><li> Aggiungere 3 ml di soluzione di LCM Additivo (fornito nel kit) al lisato e mescolare nel vortex per 5 secondi. Centrifugare per 30 secondi per raccogliere il liquido sul fondo della provetta. </li><li> Centrifugare il filtro di pre-bagnato per ~ 30 secondi alla massima velocità per rimuovere il liquido. </li><li> Aggiungere 1,25 volumi (in questo caso 129 ml) di 100% di etanolo alla miscela lisato, delicatamente vortice o pipetta su e giù. ** Questo metodo recuperare entrambe le specie di RNA grandi e piccoli. </li><li> Caricare il campione sul microfiltro pre-umidificati, e centrifugare a 10.000 xg per 1 min per legare l&#39;RNA al filtro. Quindi lavare micro filtro con 180 ml di &quot;soluzione di lavaggio 1, R21; centrifugare a 10.000 xg per 1 min. Nota: Da questo punto in tutte le centrifugazioni dovrebbe essere fatto a 13.000 xg, o la velocità massima. </li><li> Lavare Filtro due volte con 180 ml di &quot;soluzioni di lavaggio 2 e 3&quot;, rispettivamente come indicato dal protocollo del kit. Centrifugare per 30 secondi, poi scarta il flusso attraverso, e girare il filtro per 1 min per rimuovere il liquido residuo e asciugare il filtro. </li><li> Trasferire filtro per un nuovo tubo di raccolta. </li><li> Soluzione calda eluizione a -95 ° C (fornito con il kit). </li><li> Applicare 10 ml di soluzione di eluizione preriscaldato al centro del filtro, chiudere il tappo e conservare per 5 minuti a RT. Poi centrifugare per 1 minuto per eluire l&#39;RNA, e ripetere questo passaggio una volta a cedere ~ 20 ml di RNA. </li><li> Eseguire il trattamento DNasi I per 15 minuti a 37 ° C. </li><li> Quantificare l&#39;RNA da spettrofotometro con assorbanza a 260 nm. Il rapporto di densità ottica a lunghezze d&#39;onda di 260 nm e 280 nm viene utilizzato per valutare la purezzal&#39;RNA (vedi Tabella 1) 15. </li></ol> 6. Analisi di espressione genica Nota: espressione genica delle specie di RNA lunghi (come mRNA e lncRNAs) è mostrato in Fase 6.1 e specie di RNA brevi (come microRNA) è mostrata in fase 6.2. Sono disponibili diversi kit per RT-qPCR e la quantità di RNA richiesta varia a seconda del kit (non più di 10 ng). RNA analisi di sequenziamento è descritto in 6.3. <ol><li> Trascrivere inversa (RT) l&#39;RNA in cDNA mescolare 5 ml di 20 ng / mL campione di RNA con 5 ml di master mix RT (tampone RT, dNTP di, primer casuali, RT enzima inibitore RNase). Incubare la reazione per 25 minuti a 25 ° C, poi 120 min a 37 ° C e 5 min a 85 ° C. </li><li> Diluire la reazione RT 1:10 con RNasi acqua gratuita e utilizzare 2,5 microlitri per 10 microlitri reazione qPCR con primer o set di primer / sonde per i geni di interesse. Nota: Per facilitare l&#39;analisi di RNA parzialmente degradati Si consiglia di utilizzare primer for ampliconi più brevi (meno di 100 bp) 16. (Tabella 2B) <ol><li> Per l&#39;analisi di espressione dei microRNA eseguire reazione RT mescolando 5 ml di 2-10 ng / mL campione di RNA con 5 ml di master mix RT. Nota: ogni tecnologia di rilevamento microRNA disponibile in commercio a base di PCR richiede l&#39;uso di primer RT proprietari e specifici. </li></ol></li><li> In alternativa o in aggiunta a RT-PCR, utilizzare generazione sequenziamento successivo (RNA-seq o piccolo-RNA-seq) per quantificare diverse specie di RNA. Tipicamente, usare 500 ng di RNA per questa procedura (Tabella 3) ed eseguire il metodo come indicato dallo strumento costruttore e rilevamento. </li></ol>

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The authors have nothing to disclose.

Profilo di espressione genica da campioni umani può essere impegnativo, non solo per la qualità o la quantità di tessuto a disposizione, ma anche per le diverse entità istologiche presenti in un dato campione di tessuto. Ciò è particolarmente impegnativo nella prostata in cui tessuti benigni sono in gran parte i tessuti stromali e le aree di cancro sono privi di stroma. LCM facilita separazione fisica di stroma prostatico e l&#39;epitelio RNA per una firma più accurata dei due diversi tipi di cellule (Fig...

La prostata è un tessuto eterogeneo composto da epitelio secernente disposti in acini ghiandolari circondato da stroma fibromuscolare composta principalmente da muscolatura liscia 1. Il vano epiteliale è composto da cinque diversi tipi di cellule, ma organizzati: cellule basali, cellule, cellule neuroendocrine secretoria, cellule amplificazione di transito e cellule staminali 2. Nel cancro della prostata (APC), che deriva dalle cellule epiteliali luminali, la crescita del adenocarcinoma provoca un evidente declino progressivo del vano stromale 3. Per queste ragioni, campioni di tessuto saranno chiare differenze nella proporzione di stromali e cellule epiteliali tipo basato sulla misura dei PCA. Queste differenze possono portare a ipotesi di parte dei dati di espressione genica acquisiti dai tessuti interi senza riguardo per microdissezione del tipo cellulare desiderato. Pertanto, per eliminare questa tendenza è indispensabile separare tipi cellulari prima dell&#39;estrazione dell&#39;RNA eanalisi di espressione genica. Macrodissection o microdissezione possono essere utilizzati per separare fisicamente aree epiteliali ben caratterizzati dallo stroma circostante 4 -6. Macrodissection è tipicamente fatto con una lama di rasoio sotto un microscopio da dissezione e funziona bene per separare grande PCa noduli da stroma, ma non è in grado di rimuovere dell&#39;epitelio benigna da stroma circostante (vedi esempio di iperplasia prostatica istologia in figura 1). Microdissezione con un laser (LCM) è significativamente più manodopera rispetto macrodissection, ma può sezionare molto preciso epitelio benigna 4. Pubblicazioni recenti del nostro laboratorio hanno dimostrato che l&#39;RNA può essere estratto con successo da LCM da entrambi inclusi in paraffina (FFPE) biopsie fissati in formalina o tessuti congelati 4,7-9. Le principali sfide a estrazione LCM-RNA sono 1) a sezionare con precisione le aree desiderate del tessuto, e 2) per preservare RIntegrità NA durante il 4,10 LCM e isolamento del processo. RNA isolato da popolazioni cellulari pure può essere utilizzato per l&#39;analisi di espressione genica in diversi modi tra cui trascrizione inversa PCR quantitativa (RT-qPCR) 7,8, microarray 11, e profondo -sequencing 12-14. L&#39;obiettivo di questo protocollo è quello di isolare RNA totale da LCM prostatico epitelio dal tessuto congelato per le analisi di espressione genica a valle.

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

laser-capture microdissection of human prostatic epithelium for rna analysis

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

In un precedente studio abbiamo dimostrato l&#39;uso di LCM per raccogliere tessuti epiteliali e stromali confrontare profilo di espressione mediante RT-qPCR di mRNA e microRNA dai tessuti prostatici congelati e FFPE dallo stesso paziente 4. LCM richiede molto tempo, specialmente se grandi quantità di RNA è da raccogliere per l&#39;analisi di sequenziamento di prossima generazione. Pertanto, è fondamentale per mantenere lo spazio di lavoro e strumenti RNase. Si raccomanda di controllare la qualità e cellu...

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.

Laser-capture Microdissezione di Human prostatica Epitelio per l&#39;analisi di RNA

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

13.3K 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

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.

Dissezione di Human Elements corpo vitreo per l&#39;analisi proteomica

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

Ricerca

Medicina

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.

Protocollo migliorato per microdissezione laser di isole pancreatiche umane da campioni chirurgici

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.

Formazione di epitelio prostatico umano utilizzando tessuti ricombinazione dei roditori urogenitale Mesenchima Sinus e cellule staminali umane

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.

Renale Capsule xenotrapianto e sottocutanea Pellet impianto per la valutazione di carcinogenesi della prostata e dell&#39;iperplasia prostatica benigna

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.

Lo studio diabete Through the Eyes of un pesce: Microdissection, visualizzazione e analisi della tg(fli:EGFP) adulto Vasculature retinico Zebrafish

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.

Trattamento chirurgico per l'iperplasia prostatica benigna: Holmium Laser Enucleation della prostata (HoLEP).

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.

Spaziali quantificazione delle droghe nelle lesioni di tubercolosi polmonare di Laser Capture Microdissection liquido cromatografia spettrometria di massa (LCM-LC/MS)

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.

Nanoparticelle di ossido di ferro rivestite con Polyethyleneimine come un veicolo per la consegna di piccolo RNA interferente ai macrofagi In Vitro e In Vivo

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.

Isolamento di adipociti vitali e frazione vascolare stromale dal tessuto adiposo viscerale umano adatto per l'analisi dell'RNA e la fenotipizzazione dei macrofagi

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.

Trapianto di epitelio pigmentato retinico in un modello di primati non umani per malattie degenerative della retina

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