Este trabajo fue financiado por la Sociedad Canadiense del Cáncer del Instituto de Investigación, conceder ninguna. 019038.

<html> 1. Recogida de muestras de próstata <ol><li> Recoger las muestras de la próstata en el momento de la prostatectomía. La muestra se orienta utilizando puntos de referencia anatómicos. La próstata y las vesículas seminales están pintadas de la siguiente manera: verde, lado derecho, lado izquierdo azul. </li><li> Una sección media transversal al azar de la próstata se toma perpendicularmente a la superficie rectal, se congelaron en nitrógeno líquido, y se almacenó a -80 ° C 9. </li><li> Rebanadas de muestras depositadas son fotocopias, orientado (anterior, posterior, derecha e izquierda), quadrisected. Las secciones se cortan con el criostato. </li><li> Las secciones se tiñeron con H &amp; E y revisado por un patólogo para determinar y delimitar las áreas tumorales en comparación con lo normal en las diapositivas de colores y una imagen correspondiente. Las áreas marcadas son usadas como una guía para indicar las zonas de la que extraer el tejido tumoral de que el ARN se extrajo en los pasos subsiguientes. </li></ol>romper &quot;&gt; 2. Aislamiento de ARN total, incluidos el miRNA, a partir de muestras <ol><li> Colocar las muestras congeladas de próstata en hielo seco y refiriéndose a la delineada fotocopia, cortar una pequeña porción del tumor de próstata (entre 50 a 100 mg). </li><li> Homogeneizar el tejido del tumor de próstata en 1 ml de reactivo TRIzol. Las cantidades en los siguientes pasos se basan en el uso de 1 ml de reactivo TRIzol. </li></ol> Nota: Aquí hemos utilizado reactivo TRIzol para extraer ARN, sin embargo otros kits que aíslan pequeños ARN que contienen ARN total también se puede utilizar. <ol start="3"><li> Se incuban las muestras homogeneizadas durante 5 minutos a temperatura ambiente. </li><li> Añadir 0,2 ml de cloroformo para las muestras y agitar enérgicamente durante 15 segundos. Incubar las muestras durante 3 minutos a temperatura ambiente, luego se centrifuga a 12.000 xg durante 15 minutos a 4 ° C. </li><li> Transferir la fase acuosa superior incolora a tubos nuevos, y añadir 0,5 ml de alcohol isopropílico.Incubar las muestras durante 10 minutos a temperatura ambiente, luego se centrifuga a 12.000 xg durante 10 minutos a 4 ° C. </li><li> Aspirar cuidadosamente el sobrenadante sin perturbar el sedimento que contiene el ARN. Lavar el precipitado de ARN con 1 ml de etanol al 75%. Vortex la muestra y de re-sedimento por centrifugación durante 5 minutos a 7.500 xg a 4 ° C. </li><li> Aspirar cuidadosamente el sobrenadante y secar el pellet de ARN durante 5-10 minutos, asegurándose de que el precipitado de ARN no está completamente seca. Vuelva a disolver en agua libre de nucleasa apropiado para el tamaño de pellets. Medir la concentración de ARN utilizando el espectrofotómetro NanoDrop 1000 (absorbancia medida a 260 nm y 280 nm). </li><li> Compruebe la calidad y la integridad de las muestras de ARN utilizando Agilent Bioanalyzer. </li></ol> 3. La transcripción inversa del ARN <ol><li> La transcripción inversa del ARN se realizó utilizando el Kit de miScript transcripción reversa de acuerdo a las instrucciones del fabricante (Qiagen). Este kit incluye untranscriptasa inversa y una polimerasa poli (A). El tampón miScript RT incluye Mg 2 +, dNTPs, oligo-dT primers y cebadores aleatorios. </li><li> Utilizar entre 10 pg y 1 g de ARN para sintetizar ADNc. Si utiliza más de 1 mg de ARN, ampliar la reacción linealmente con el volumen apropiado. </li><li> Prepare una mezcla maestra que contiene 5 veces miScript RT Buffer (4 l), Reverse miScript Mix transcripción (1 l), y el agua libre de ARNasa para que las reacciones a volumen final de 20 l. También se incluyen plantillas de ARN (hasta 1 g) en la mezcla maestra. </li><li> Se incuban las muestras durante 60 minutos a 37 ° C, seguido inmediatamente por una incubación durante 5 minutos a 95 ° C. Este paso puede realizarse en una máquina de PCR, calentando baño bloque, o agua. Termocicladores son el método más adecuado y preciso. Guarde el cDNA en el hielo para el corto plazo, y -20 ° C para el almacenamiento a largo plazo. </li></ol> 4. Generación de una curva estándar <ol><li> Antes de experperimento con miARNs diana, una curva estándar se genera mediante el uso de cDNAs de concentraciones conocidas en contra de sus puntos de cruce (CP) (Figura 2). </li><li> Preparar una serie de diluciones de dos veces, 10 veces, 50 veces, 250-veces, y 1250-pliegue del cDNA original de una muestra que se sabe que tiene una expresión sustancial de su gen de interés. </li><li> Ejecute el PCR como se especifica en la Sección 5 &quot;en tiempo real PCR para la detección de miRNA&quot;, con la salvedad de que ADNc está en diluciones en serie no es una dilución 40x estática. </li><li> Realizar un análisis utilizando el software de RelQuant (Roche) para generar la curva estándar. </li></ol> Nota: una nueva curva estándar se debe generar para cada gen de interés. 5. PCR en tiempo real para la detección de miRNA <ol><li> PCR en tiempo real de miRNAs se ha realizado mediante miScript SYBR Green PCR kit de ensayo y miScript Primer acuerdo a las instrucciones del fabricante (Qiagen). Prepare una mezcla maestra que contenía2x QuantiTect SYBR Green PCR Master Mix, 10x Primer miScript Universal, ensayo 10x Primer miScript, y el agua libre de RNasa. Prepare una mezcla maestra para una reacción de volumen de 20 l. </li><li> El Ensayo de imprimación es específica para el miARN de interés. Para reconstituir 10x Ensayo imprimación miScript, centrifugar el vial brevemente, y añadir 550 l de tampón TE, pH 8,0. Vortex el vial brevemente para mezclar, cebadores alícuotas a volúmenes más pequeños, y almacenar a -20 ° C. Dos cebadores se requiere:. Cebadores para el gen diana y el gen de referencia RNU6B usedas es el gen de referencia. </li><li> Diluir las alícuotas de cDNA adicionales 40x y almacenar a -20 ° C. </li><li> ADNc sirve como molde para la PCR. Utilice 2 l de cDNA diluido 40x y dispensar a los 20 l luz de ciclo, los capilares (Roche). </li><li> Añadir 18 l de la mezcla maestra a cada capilar, y centrifugar utilizando un adaptador capilar. </li><li> Coloque los vasos capilares en un capilar basada en tiempo real termociclador, como LightCycler 3.5 Real-Time PCR System con un formato de carrusel 32-capilar. </li><li> Ejecutar el programa de ciclos de PCR como sigue: Para activar la polimerasa HotStarTaq que está en el 2x QuantiTect SYBR Green PCR Master Mix, pre-incubar a 95 ° C durante 15 minutos. Seguido por 50 ciclos de: La desnaturalización, 15 s, 94 ° C; Recocido, 30 s, 55 ° C; Extensión, 30 s, 70 ° C. </li><li> Selección de una muestra para ser el calibrador, y establecer su importe objetivo normalizado a 1. Comparar la expresión relativa de los genes miARN en todas las otras muestras en el calibrador. </li></ol> Nota: En un estudio, la misma muestra de calibración se debe utilizar para mantener la consistencia de los resultados. 6. Análisis de datos <ol><li> Curvas de amplificación de la reacción de PCR se representan de forma gráfica y numérica por la versión del software Molecular bioquímicos LightCycler 3.5 (Roche). Cuantificar las reacciones en la &quot;cuantificación&quot; ficha, unaª exportar los datos a un archivo de texto. </li><li> Importar los datos al software de análisis RelQuant (Roche) para generar resultados de la cuantificación. Importación de archivos separados para gen diana, el gen de referencia, y los datos estándar de la curva. </li><li> Especifique la posición del calibrador para tanto el objetivo como gen de referencia. También debe especificar las posiciones de las muestras. Los datos se expresan como el objetivo para hacer referencia a relación de las diferentes muestras, dividido por el objetivo de proporción de referencia del calibrador. La curva estándar generada previamente por un miRNA particular y limpieza de genes se utiliza como patrón de referencia para la extrapolación de los datos cuantitativos de los genes miARN objetivos de concentraciones desconocidas. </li><li> Tres repeticiones de muestras son analizadas como un grupo y las concentraciones medias y desviaciones estándar de la triplicado, se calcula. Si uno de los triplicados es incompatible con el resto del conjunto, se excluyó por el programa. </li></ol> 7. Los resultados representativos &lt;clase p = &quot;jove_content&quot;&gt; Un ejemplo de qPCR análisis sobre muestras de próstata se muestra en la Figura 3. Los resultados se representan numéricamente, así como gráficamente. Los gráficos que muestran los niveles de expresión del gen de referencia, U6, comenzar la amplificación exponencial a aproximadamente ciclo 20, mientras que la expresión del gen diana, MIR-98, mostró una amplificación retraso aproximadamente en el ciclo 25. Los datos de este experimento se exporta como archivo de texto y analizadas por el software de análisis RelQuant. Las posiciones de los capilares que contienen el calibrador y las muestras se especifican. Figura 4 ilustra cómo el calibrador está dispuesto a ser 1, y la expresión de otras muestras relativas al calibrador. <img src="/files/ftp_upload/3874/3874fig1.jpg" alt="Figura 1"/> Figura 1. Varios pasos en miScript transcripción inversa y PCR en tiempo real. <img src="/files/ftp_upload/3874/3874fig2.jpg" alt="Figura 2"/> Figura 2. Una curva estándar se genera mediante una serie de diluciones de dos veces, 10 veces, 50 veces, 250-veces, y 1250-pliegue del cDNA muestra original. <img src="/files/ftp_upload/3874/3874fig3.jpg" alt="Figura 3"/> Figura 3. Roche Molecular bioquímicos LightCycler Software muestra la información completa de la experiencia gráfica y de texto. Cuantitativa en tiempo real las parcelas de amplificación de PCR muestran un aumento en la fluorescencia de las muestras diferentes. <img src="/files/ftp_upload/3874/3874fig4.jpg" alt="Figura 4"/> Datos de la Figura 4. Se cuantificaron utilizando el software de análisis de RelQuant LightCycler. Por lo general, tres réplicas de las muestras se analizan como un grupo y las muestras que producen resultados claramente inconsistentes son excluidos y las concentraciones medias y desviaciones estándar de la triplicado, se calcula.

<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>

No hay conflictos de interés declarado.

Expresiones aberrantes de algunos miRNAs han sido encontrados en los tumores de próstata en comparación con el tejido normal de 10, y algunos de estos miRNAs han sido mencionados como potenciales nuevos agentes terapéuticos contra el cáncer de próstata 11. Por lo tanto los niveles de expresión de miRNAs aberrantes pueden ser útiles biomarcadores de diagnóstico y / o pronóstico. La metodología qPCR en tiempo real se presenta aquí proporciona un ensayo para la cuantificación exacta de los...

<table border="1"><tbody><tr><td> Nombre del reactivo </td><td> Empresa </td><td> Número de catálogo </td></tr><tr><td> Reactivo TRIzol </td><td> Invitrogen </td><td> 15596 </td></tr><tr><td> miScript Kit de transcripción reversa </td><td> Qiagen </td><td> 218061 </td></tr><tr><td> Los ensayos miScript Primer </td><td> Qiagen </td><td> Experimento específica </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 capilares </td><td> Roche </td><td> 04929292001 </td></tr><tr><td> NanoDrop 1000 espectrofotómetro </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)

Los microARN (miRNA) son de cadena sencilla, 18-24 nucleótidos de largo, no codificantes moléculas de ARN. Ellos están involucrados en prácticamente todos los procesos celulares incluyendo el desarrollo de 1, 2 la apoptosis y la regulación del ciclo celular 3. MiRNAs se estima que regulan la expresión de 30% a 90% de los genes humanos 4 mediante la unión a sus ARNs diana mensajero (ARNm) 5. La desregulación generalizada de los miRNAs se ha informado en diversas enfermedades y subtipos de cáncer 6. Debido a su prevalencia y la estructura única, estas pequeñas moléculas tienden a ser la próxima generación de biomarcadores, agentes terapéuticos y / o objetivos. Los métodos utilizados para investigar la expresión de miRNA son SYBR Green I con base de tinte, así como Taqman-qPCR basado en la sonda. Si los miRNAs se utiliza con eficacia en el ámbito clínico, es imprescindible que su detección en muestras clínicas frescas y / o archivados ser exacto, reproducible, y specific. qPCR ha sido ampliamente utilizado para la validación de expresión de miRNAs en los análisis de todo el genoma, tales como estudios de microarrays 7. Las muestras utilizadas en este protocolo fueron de pacientes que se sometieron a prostatectomía radical por cáncer de próstata clínicamente localizado, sin embargo otros tejidos y líneas celulares se pueden sustituir in biopsias de próstata fueron broche de congelados en nitrógeno líquido después de la resección. Las variables clínicas y la información de seguimiento para cada paciente se recogieron para su análisis posterior 8. La cuantificación de los niveles de miRNA en muestras de tumores de próstata. Los pasos principales en el análisis de qPCR de los tumores son: extracción de RNA total, síntesis de ADNc, y la detección de productos específicos qPCR utilizando miRNA-cebadores. El ARN total, que incluye mRNA, Mirna, y otros pequeños RNAs fueron extraídos de las muestras con el reactivo TRIzol. Sistema de Qiagen miScript fue utilizado para sintetizar cDNA y realizar qPCR (Figura 1). MiRNAs endógenos no son polyadenylated, tanto durante el proceso de transcripción inversa, un poli (A) polimerasa polyadenylates el miARN. El miARN se utiliza como una plantilla para sintetizar cDNA utilizando oligo-dT y la transcriptasa inversa. Una secuencia de etiqueta universal en el extremo 5 &#39;de los cebadores de oligo-dT facilita la amplificación de cDNA en el paso de PCR. Amplificación por PCR producto es detectado por el nivel de fluorescencia emitida por SYBR Green, un colorante que se intercala en el ADN de doble cadena. Cebadores específicos miARN, junto con una imprimación universal que se une a la secuencia de etiqueta universal amplificar secuencias específicas miARN. Los ensayos Primer miScript están disponibles para más de un millar de miRNAs específicos de los humanos, y cientos de ratón específicos de miRNAs. Método de cuantificación relativa se utiliza aquí para cuantificar la expresión de miRNAs. Para corregir la variabilidad entre las diferentes muestras, los niveles de expresión de un miARN objetivo se normaliza a los niveles de expresión de un gen de referencia. La elección de un GEne en que para normalizar la expresión de los objetivos es crítico en el método de cuantificación relativa de análisis. Ejemplos de genes de referencia típicamente utilizados en esta capacidad son la RNU6B ARN pequeño, RNU44, y RNU48 ya que se consideran para ser expresado de forma estable en la mayoría de las muestras. En este protocolo, RNU6B se utiliza como el gen de referencia.

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

Cuantitativa de la polimerasa en tiempo real de reacción en cadena (PCR cuantitativa) es un método rápido y sensible para investigar los niveles de expresión de microARN (miRNA varias) moléculas en las muestras tumorales. El uso de este método de expresión de cientos de moléculas de diferentes miARN puede ser amplificada, cuantificado y analizado desde la misma plantilla de cDNA.

Detección de microARN en tumores de próstata por el cuantitativo PCR en tiempo real (qPCR)

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

microrna-detection-prostate-tumors-quantitative-real-time-pcr

Research

JoVE Journal

Medicine

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

24.4K vistas.  University of Toronto. Cuantitativa de la polimerasa en tiempo real de reacción en cadena (PCR cuantitativa) es un método rápido y sensible para investigar los niveles de expresión de microARN (miRNA varias) moléculas en las muestras tumorales. El uso de este método de expresión de cientos de moléculas de diferentes miARN puede ser amplificada, cuantificado y analizado desde la misma plantilla de 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.

Multiespectrales en tiempo real imágenes de fluorescencia para la detección intraoperatoria del ganglio centinela en Oncología Ginecológica

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 ...

Investigación

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.

A tiempo real Técnica de Impedancia Eléctrica base para medir la invasión de monocapa de células endoteliales de las células cancerosas

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.

Evaluación de la absorción de nanopartículas en los tumores en tiempo real utilizando intravital imagen

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.

Detección y Genogrouping de norovirus en las heces de los niños por TaqMan en un solo paso 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.

Mapeo de la heterogeneidad de la expresión de proteínas en los tumores utilizando inmunofluorescencia cuantitativa

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

Identificación de los<em&gt; La Bella Durmiente</em&gt; Transposón inserciones en tumores sólidos utilizando vinculador mediada por 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.

Movimiento de Reconversión utilizando información en tiempo real del rendimiento

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.

Usando cuantitativa en tiempo real PCR para determinar quimerismo de células donantes en un modelo competitivo Trasplante de médula ósea murina

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.

Líquido cefalorraquídeo MicroRNA perfiles utilizando cuantitativa en tiempo real 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.

En tiempo real Ensayos de citotoxicidad en sangre entera humana

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