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&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 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

Universidad de Cartagena UDEC

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.

Rilevamento MicroRNA in tumori della prostata dal 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 ...

Ricerca

Medicina

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.

Sequenziamento di piccoli RNA non codificanti da tessuti fissati in formalina ed esosomi derivati dal siero da pazienti affetti da cancro alla prostata resistenti alla castrazione

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

Ricerca sul cancro

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.

Microarray identificazione dei singoli HERV Loci Expression: Domanda di Biomarker Discovery in Cancro alla prostata

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.

Microdissezione laser di cattura della cartilagine embrionale del topo e dell'osso per l'analisi dell'espressione genica

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

Biologia dello sviluppo

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).

Analisi miRNA nel tumore alla prostata tessuti cliniche

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.

Ottimizzazione di Laser Capture Microdissection per l'isolamento dei gangli enterici dal tessuto umano fresco congelato

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

Neuroscienze

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.

RNA isolamento da cellulari sottopopolazioni specifiche utilizzando laser-capture Microdissection combinazione con Rapid immunomarcatura

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

Biologia

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.

Microdissezione laser di elevata purezza trabecolato dai Mouse occhi per analisi dell'espressione genica

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.

Un protocollo di microdissezione di cattura laser che produce RNA di alta qualità da ossa di mouse fresh-congelate

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.

Applicazione della microdisezione laser per scoprire la trascritomica regionale nel tessuto renale umano

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

Laser-capture Microdissezione di Human prostatica Epitelio per l'analisi di RNA

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