Questo lavoro è stato finanziato dalla Canadian Cancer Society Research Institute, concedere no. 019038.

<html> 1. Prostata Raccolta dei campioni <ol><li> Raccogliere i campioni prostata al momento della prostatectomia. Il campione è orientato con punti di repere anatomici. La prostata e delle vescicole seminali sono dipinti come segue: verde lato destro, blu lato sinistro. </li><li> Una sezione centrale casuale trasversale della prostata è presa perpendicolarmente alla superficie rettale, congelato in azoto liquido e conservati a -80 ° C 9. </li><li> Fette da banche di campioni sono fotocopiato, orientata (anteriore, posteriore, destro e sinistro), quadrisected. Le sezioni sono tagliati con il criostato. </li><li> Le sezioni sono colorate con H &amp; E e rivisto da un patologo per determinare e delineare le aree tumorali rispetto al normale sui vetrini colorati e una corrispondente immagine. Le aree marcate vengono utilizzati come guida per indicare le aree da cui estrarre il tessuto tumorale da cui RNA sarà estratto nelle fasi successive. </li></ol>rompere &quot;&gt; 2. Isolamento RNA totale, con miRNA, da Campioni <ol><li> I campioni prostata Posizionare congelati su ghiaccio secco e riferendosi alla delineato fotocopia, taglio di una piccola porzione del tumore della prostata (tra 50 e 100 mg). </li><li> Omogeneizzare il tessuto del tumore della prostata in 1 ml di reagente TRIzol. Le quantità nei passaggi seguenti si basano sull&#39;uso di 1 ml di reagente TRIzol. </li></ol> Nota: Qui abbiamo utilizzato reagente Trizol RNA per estrazione, tuttavia altri kit che isolano piccoli RNA contenenti RNA totale può anche essere usato. <ol start="3"><li> Incubare i campioni omogeneizzati per 5 minuti a temperatura ambiente. </li><li> Aggiungere 0,2 mL di cloroformio per i campioni e agitare vigorosamente per 15 secondi. Incubare campioni per 3 minuti a temperatura ambiente, quindi si centrifuga a 12.000 xg per 15 minuti a 4 ° C. </li><li> Trasferire la incolore fase acquosa superiore ai tubi freschi, e aggiungere 0,5 ml di alcool isopropilico.Incubare campioni per 10 minuti a temperatura ambiente, quindi si centrifuga a 12.000 xg per 10 minuti a 4 ° C. </li><li> Aspirare accuratamente il sopranatante senza disturbare il pellet contenente l&#39;RNA. Lavare il pellet di RNA con 1 mL di etanolo al 75%. Vortex il campione e ri-sedimento mediante centrifugazione per 5 minuti a 7.500 xg a 4 ° C. </li><li> Aspirare accuratamente la supernatante e seccare il pellet di RNA per 5-10 minuti, assicurandosi che il pellet di RNA non è completamente asciutto. Re-sciogliere in acqua priva di nucleasi opportuno dimensione delle pastiglie. Misurare la concentrazione di RNA utilizzando lo spettrofotometro NanoDrop 1000 (misura assorbanza a 260 nm e 280 nm). </li><li> Controllare la qualità e l&#39;integrità dei campioni di RNA usando Agilent Bioanalyzer. </li></ol> 3. Trascrizione inversa di RNA <ol><li> Trascrizione inversa di RNA è stata effettuata utilizzando miScript Kit trascrizione inversa secondo le istruzioni del produttore (Qiagen). Questo kit comprende untrascrittasi inversa e un (A) poli polimerasi. Il tampone miScript RT include Mg 2 +, dNTPs, oligo-dT primer e primer casuali. </li><li> Utilizzare tra 10 e 1 pg pg di RNA per sintetizzare cDNA. Se si utilizza più di 1 mg di RNA, la reazione scalare linearmente al volume adeguato. </li><li> Preparare una master mix che contiene 5X miScript RT Buffer (4 pl), Mix Reverse miScript Trascrizione (1 pl), e RNasi-free acqua per portare le reazioni al volume finale di 20 microlitri. Includere anche RNA modello (fino a 1 mg) nel mix master. </li><li> Incubare i campioni per 60 minuti a 37 ° C seguita immediatamente da una incubazione per 5 minuti a 95 ° C. Questa fase può essere eseguita in una macchina PCR, riscaldamento bagno blocco, o acqua. Termociclatori sono il metodo più adeguato e preciso. Conservare il cDNA su ghiaccio per breve termine, e -20 ° C per una conservazione a lungo termine. </li></ol> 4. Generazione di una curva standard <ol><li> Prima esperiiment con miRNA bersaglio, una curva standard è generata usando cDNA di concentrazioni note contro i loro punti di incrocio (CP) (Figura 2). </li><li> Preparare una serie di diluizioni di 2 volte, 10-volte, 50-volte, 250-volte, e 1250 volte il cDNA originale di un campione che è noto per avere una espressione sostanziale del gene di interesse. </li><li> Eseguire il PCR, come specificato nella sezione 5 &quot;Real-time PCR per il rilevamento di miRNA&quot;, con la modifica che è cDNA in diluizioni seriali non una diluizione statica 40x. </li><li> Eseguire l&#39;analisi utilizzando il software RelQuant (Roche) per generare la curva standard. </li></ol> Nota: una nuova curva standard deve essere generata per ogni gene di interesse. 5. Real-time PCR per il rilevamento di miRNA <ol><li> Real time PCR per miRNA è stata eseguita utilizzando miScript SYBR Green PCR Assay Kit e Primer miScript secondo le istruzioni del produttore (Qiagen). Preparare una master mix contenente2x QuantiTect SYBR Green PCR Master Mix, 10x Primer miScript Universal, 10x Assay Primer miScript e acqua priva di RNasi. Preparare una master mix per una reazione di un volume di 20 microlitri. </li><li> Il saggio Primer è specifico per il miRNA di interesse. Per Assay miScript 10x Primer ricostituire, centrifugare le provette brevemente e aggiungere 550 microlitri TE, pH 8,0. Vortex il flaconcino brevemente per miscelare, primer aliquote a volumi più piccoli, e conservare a -20 ° C. Due primer sono richieste:. Primer per il gene bersaglio e il gene di riferimento RNU6B è utilizzato così come il gene di riferimento. </li><li> Diluire le aliquote di cDNA 40x e conservare supplementari a -20 ° C. </li><li> serve cDNA come stampo per la PCR. Utilizzare 2 microlitri di 40x cDNA diluito e dispensiamo ai 20 pl luce cycler capillari (Roche). </li><li> Aggiungere 18 microlitri della master mix per ogni capillare, e centrifugare utilizzando un adattatore capillare. </li><li> Posizionare i capillari in un capillare basato Real-Time cycler, come LightCycler 3.5 Real-Time PCR System con un 32-capillare formato giostra. </li><li> Eseguire il programma dei cicli di PCR come segue: Per attivare polimerasi HotStarTaq che è in 2x QuantiTect SYBR Green PCR Master Mix, pre-incubare a 95 ° C per 15 minuti. Seguito da 50 cicli di: Denaturazione, 15 s, 94 ° C; Ricottura, 30 s, 55 ° C; Extension, 30 s, 70 ° C. </li><li> Selezionare un campione di essere il calibratore, e impostare il suo valore normalizzato obiettivo a 1. Confrontare l&#39;espressione relativa del miRNA in tutti gli altri campioni al calibratore. </li></ol> Nota: In uno studio, lo stesso campione di taratura deve essere utilizzato per mantenere la consistenza dei risultati. 6. Analisi dei dati <ol><li> Curve di amplificazione per le reazioni PCR sono raffigurati graficamente e numericamente da Molecular Biochemicals versione software LightCycler 3,5 (Roche). Quantificare le reazioni nella scheda &quot;quantificazione&quot;, unnd esportare i dati in un file di testo. </li><li> Importare i dati al software di analisi RelQuant (Roche) per generare risultati quantificazione. Importare file separati per gene bersaglio, gene di riferimento, e dei dati della curva standard. </li><li> Specificare la posizione del calibratore per bersaglio e gene di riferimento. Anche specificare le posizioni dei campioni. I dati sono espressi come la destinazione di riferimento rapporto di diversi campioni diviso per il bersaglio rapporto riferimento del calibratore. La curva standard precedentemente generato per un miRNA particolare e gene housekeeping è usato come standard di riferimento per estrapolare i dati quantitativi per gli obiettivi miRNA di concentrazioni sconosciute. </li><li> Tre repliche di campioni vengono analizzati come gruppo e le concentrazioni medie e le deviazioni standard del triplice copia viene calcolato. Se uno dei triplicati è incompatibile con il resto del set, verrà escluso dal programma. </li></ol> 7. Risultati rappresentativi &lt;class p = &quot;jove_content&quot;&gt; Un esempio di analisi su campioni qPCR prostata è mostrato in Figura 3. I risultati sono rappresentati numericamente, così come graficamente. I grafici che mostrano i livelli di espressione del gene di riferimento, U6, iniziano amplificazione esponenziale a circa ciclo 20, mentre l&#39;espressione del gene bersaglio, miR-98, mostrato amplificazione ritardato circa al ciclo 25. I dati di questo esperimento è stato esportato come file di testo e analizzati dal software di analisi RelQuant. Posizioni del capillari contenenti il calibratore ei campioni sono specificati. Figura 4 illustra come il calibratore è impostato a 1, e l&#39;espressione di altri campioni relativi al calibratore. <img src="/files/ftp_upload/3874/3874fig1.jpg" alt="Figura 1"/> Figura 1. Varie fasi miScript trascrizione inversa e Real-time PCR. <img src="/files/ftp_upload/3874/3874fig2.jpg" alt="Figura 2"/> Figura 2. Una curva standard è generata usando una serie di diluizioni di 2 volte, 10-volte, 50-volte, 250-volte, e 1250 volte il campione originale cDNA. <img src="/files/ftp_upload/3874/3874fig3.jpg" alt="Figura 3"/> Figura 3. Roche Molecular LightCycler Biochemicals Software mostra le informazioni di tutto l&#39;esperimento graficamente e dal testo. Quantitativi in ​​tempo reale grafici di amplificazione PCR mostrano un aumento della fluorescenza da campioni diversi. <img src="/files/ftp_upload/3874/3874fig4.jpg" alt="Figura 4"/> I dati Figura 4. Sono stati quantificati utilizzando RelQuant software di analisi LightCycler. Di solito, tre repliche di campioni vengono analizzati come un gruppo di campioni e che producono risultati chiaramente incoerenti sono escluse e le concentrazioni medie e le deviazioni standard del triplice copia viene calcolato.

<ol>
	<li>Reinhart, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+plants.">MicroRNAs in plants.</a> Genes Dev. 16, 1616-1616 (2002).</li><li>Xu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+microRNA+Mir-14+suppresses+cell+death+and+is+required+for+normal+fat+metabolism.">The Drosophila microRNA Mir-14 suppresses cell death and is required for normal fat metabolism.</a> Curr. Biol. 13, 790 (2003).</li><li>Bueno, M. J., Perez, d. e. C. a. s. t. r. o., I, ., Malumbres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+proliferation+pathways+by+microRNAs.">Control of cell proliferation pathways by microRNAs.</a> Cell Cycle. 7, 3143-3143 (2008).</li><li>Friedman, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92 (2009).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116, 281-281 (2004).</li><li>Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+consequences+of+microRNA+dysregulation+in+cancer.">Causes and consequences of microRNA dysregulation in cancer.</a> Nat. Rev. Genet. 10, 704 (2009).</li><li>Coppola, V., De, M. R., Bonci, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+and+prostate+cancer.">MicroRNAs and prostate cancer.</a> Endocr. Relat. Cancer. 17, 1-1 (2010).</li><li>Gordanpour, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-221+Is+down-regulated+in+TMPRSS2:ERG+fusion-positive+prostate+cancer.">miR-221 Is down-regulated in TMPRSS2:ERG fusion-positive prostate cancer.</a> Anticancer Res. 31, 403-403 (2011).</li><li>Nam, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+TMPRSS2:ERG+fusion+gene+predicts+cancer+recurrence+after+surgery+for+localised+prostate+cancer.">Expression of the TMPRSS2:ERG fusion gene predicts cancer recurrence after surgery for localised prostate cancer.</a> Br. J. Cancer. 97, 1690 (2007).</li><li>Ambs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+profiling+of+microRNA+and+messenger+RNA+reveals+deregulated+microRNA+expression+in+prostate+cancer.">Genomic profiling of microRNA and messenger RNA reveals deregulated microRNA expression in prostate cancer.</a> Cancer Res. 68, 6162 (2008).</li><li>Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+miR-34a+inhibits+prostate+cancer+stem+cells+and+metastasis+by+directly+repressing+CD44.">The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44.</a> Nat. Med. 17, 211 (2011).</li></ol>

Non ci sono conflitti di interesse dichiarati.

Espressioni aberranti di alcune miRNA sono stati costantemente nei tumori della prostata rispetto al tessuto normale 10, e alcuni di questi miRNA sono stati nominati come potenziali agenti terapeutici nuovi contro il cancro della prostata 11. Quindi i livelli di espressione dei miRNA aberranti possono essere utili biomarcatori diagnostici e / o prognostico. The Real-Time qPCR metodologia qui presentata prevede un test per la quantificazione precisa dei livelli di miRNA nei tessuti tumorali della pr...

<table border="1"><tbody><tr><td> Nome del reattivo </td><td> Azienda </td><td> Numero di catalogo </td></tr><tr><td> Reagente TRIzol </td><td> Invitrogen </td><td> 15596 </td></tr><tr><td> miScript Kit trascrizione inversa </td><td> Qiagen </td><td> 218061 </td></tr><tr><td> Saggi Primer miScript </td><td> Qiagen </td><td> Esperimento specifica </td></tr><tr><td> miScript SYBR Green PCR Kit </td><td> Qiagen </td><td> 218073 </td></tr><tr><td> LightCycler 3,5 Real-Time PCR System </td><td> Roche </td><td></td></tr><tr><td> Light Cycler Capillari </td><td> Roche </td><td> 04929292001 </td></tr><tr><td> NanoDrop 1000 spettrofotometro </td><td> Thermo Scientific </td><td> 2538 </td></tr><tr><td> Agilent 2100 Bioanalyzer </td><td> G2943CA </td><td></td></tr></tbody></table>

microrna detection in prostate tumors by quantitative real-time pcr (qpcr)

I microRNA (miRNA) sono a singolo filamento, lunghe 18-24 nucleotidi, non codificanti molecole di RNA. Essi sono coinvolti praticamente in ogni processo cellulare, compreso lo sviluppo 1, apoptosi 2 e regolazione del ciclo cellulare 3. MicroRNA sono stimati per regolare l&#39;espressione del 30% al 90% dei geni umani 4 legandosi al RNA bersaglio loro messaggero (mRNA) 5. La diffusa disregolazione dei miRNA è stata segnalata in varie malattie e sottotipi di cancro 6. Grazie alla loro prevalenza e la struttura unica, queste piccole molecole sono suscettibili di essere la prossima generazione di biomarcatori, agenti terapeutici e / o obiettivi. I metodi utilizzati per indagare l&#39;espressione miRNA sono SYBR green I dye-based così come Taqman-probe qPCR based. Se miRNA devono essere efficacemente utilizzato in ambito clinico, è indispensabile che la loro rilevazione in campioni clinici freschi e / o archiviati essere precisa, riproducibile e specific. qPCR è stato ampiamente utilizzato per validare l&#39;espressione dei miRNA nelle analisi dell&#39;intero genoma, quali studi di microarray 7. I campioni utilizzati in questo protocollo provenivano da pazienti sottoposti a prostatectomia radicale per tumore della prostata clinicamente localizzato, ma in altri tessuti e linee cellulari possono essere sostituiti dentro i campioni della prostata sono stati snap-congelato in azoto liquido dopo la resezione. Le variabili cliniche e follow-up di informazione per ogni paziente sono stati raccolti per la successiva analisi 8. Quantificazione dei livelli di miRNA in campioni tumorali della prostata. Le fasi principali nell&#39;analisi qPCR dei tumori sono: estrazione di RNA totale, sintesi di cDNA, e la rilevazione dei prodotti qPCR miRNA utilizzando primer specifici. L&#39;RNA totale, che include mRNA, miRNA, e altri piccoli RNA sono stati estratti da campioni usando il reagente TRIzol. Sistema miScript Qiagen è stato usato per sintetizzare cDNA ed eseguire qPCR (Figura 1). MiRNA endogeni non sono polyadenylated, quindi durante il processo di trascrizione inversa, un poli (A) polimerasi polyadenylates il miRNA. Il miRNA viene utilizzato come modello per sintetizzare cDNA utilizzando oligo-dT e trascrittasi inversa. Una sequenza tag universale della estremità 5 &#39;del primer oligo-dT facilita l&#39;amplificazione del cDNA in fase di PCR. Prodotto di amplificazione PCR viene rilevato dal livello di fluorescenza emessa da SYBR Green, un colorante intercalante che in DNA a doppio filamento. Primer specifici miRNA, insieme con un Primer universale che si lega alla sequenza di tag universale amplificare specifiche sequenze di miRNA. I saggi Primer miScript sono disponibili per oltre un migliaio di umano-specifici microRNA, e centinaia di murina specifici miRNA. Metodo di quantificazione relativa è qui usato per quantificare l&#39;espressione dei miRNA. Per correggere la variabilità tra campioni differenti, i livelli di espressione di un miRNA bersaglio viene normalizzato i livelli di espressione di un gene di riferimento. La scelta di un gene su cui normalizzare l&#39;espressione di bersagli è critica nel metodo di quantificazione relativa analisi. Esempi di geni di riferimento tipicamente usati in questa capacità sono RNU6B piccolo RNA, RNU44, e RNU48 come sono considerati essere stabilmente espressa nella maggior parte dei campioni. In questo protocollo, RNU6B viene utilizzato come il gene di riferimento.

Watch this Scientific Journal Video about MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR at JoVE.com

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

Quantitativa reale tempo di reazione a catena della polimerasi (qPCR) è un metodo rapido e sensibile per indagare i livelli di espressione di microRNA (miRNA vari) molecole in campioni tumorali. Usando questo metodo di espressione di centinaia di molecole diverse miRNA possono essere amplificati, quantificati e analizzati utilizzando lo stesso modello cDNA.

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 ...

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Research

JoVE Journal

Medicine

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

24.4K Views.  University of Toronto. Quantitativa reale tempo di reazione a catena della polimerasi (qPCR) è un metodo rapido e sensibile per indagare i livelli di espressione di microRNA (miRNA vari) molecole in campioni tumorali. Usando questo metodo di espressione di centinaia di molecole diverse miRNA possono essere amplificati, quantificati e analizzati utilizzando lo stesso modello cDNA.

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

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

Multispettrali in tempo reale immagini a fluorescenza per il rilevamento intraoperatoria del linfonodo sentinella in Oncologia Ginecologica

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

Ricerca

Medicina

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

Un tempo reale impedenza elettrica tecnica basata su Misura Invasione di cellule endoteliali monostrato dalle cellule tumorali

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

Valutazione della captazione nanoparticelle nei tumori in tempo reale utilizzando intravitale Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

Detection and Genogrouping of Noroviruses from Children&#039;s Stools By Taqman One-step RT-PCR

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

Rilevamento e Genogrouping di Norovirus dalle feci dei bambini By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

Mappatura eterogeneità di espressione delle proteine ​​nei tumori mediante immunofluorescenza quantitativa

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

Identificazione dei<em&gt; Bella Addormentata</em&gt; Inserimenti trasposone nei tumori solidi utilizzando Linker-mediata PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

Movimento Riqualificazione con feedback in tempo reale delle prestazioni

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

Utilizzando quantitativa real-time PCR per la determinazione cellulare attecchimento dei donatori in un modello murino competitivo trapianto di midollo osseo

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

Liquido cerebrospinale MicroRNA profiling Utilizzando quantitativa Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

In tempo reale citotossicità saggi in Human Sangue intero

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with ...