Name: clinicopathological analysis of mirna expression in breast cancer tissues by using mirna in situ hybridization
Uploaded: 2016-06-07T15:00:00.000+00:00
Duration: 6 min 1 s
Description: Watch this Scientific Journal Video about Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization at JoVE.com

This work was supported by the NIH grant (5R01 CA178386-03) and the USC ASPIRE-1 grant to HC. We would like to thank Vrushab Gowda for his assistance with manuscript.

Este estudo foi aprovado pelo Institutional Review Board do Hospital Chonnam National University Hwasun e University of South Carolina. amostras TMA compostas de câncer de mama e tecidos mamários normais adjacentes combinados foram fornecidos pelo Biobank de Chonnam National Hwasun Hospital Universitário, um membro da Coreia do Biobank rede. 1. Preparação de Soluções <ol><li> Prepare 1 litro de DNase e RNase água livre. Faça todas as soluções químicas usando dietilpirocarbonato (DEPC) água tratada. Tratar 1 L de água com 500 ul de DEPC e incubar durante 3 horas a temperatura ambiente. Autoclave a água para inactivar DEPC. Cuidado: Não inalar DEPC, uma vez que é conhecido agente cancerígeno. </li><li> Prepare 1x PBS a partir de 10x PBS usando DEPC água tratada. </li><li> Prepare 20x SSC (dissolver 175,3 g de NaCl e 88,2 g de citrato de sódio em 800 ml de água, ajustar o pH a 7,0 com algumas gota de 14 N solução de HCl e ajustar o volume a 1 litro com water), paraformaldeído a 4%, etanol a 95%, etanol a 80% usando água tratada com DEPC. </li><li> Adicionar 8 mL de trietanolamina e 1,05 mL de HCl em 590 ml de água tratada com DEPC e adicionar 1,5 ml de anidrido acético para acetilação. </li><li> Preparar 50 ug / mL de proteinase K em tampão (Tris-HCl a 50, pH 8,0 em água tratada com DEPC). </li><li> Preparar tampão de hibridação constituída por 500 mL de formamida, 250 pL de 20x SSC, 100 ul de solução 50x de Denhardt, 12,5 ul de t-RNA, 2,5 ul de ADN de esperma de arenque, 30 ul de RVC, 0,02 g de reagente de bloqueio e 50 ul de água DEPC. Prepare fresco. </li></ol> 2. Os pontos tecido mamário <ol><li> Corte de 5 mm secções embebidas em parafina fixadas em formalina com micrótomo e aplicar a secção de cobrar slide 14. Nota: O tecido da mama usada aqui foi previamente preparado a partir da Coreia do Biobank rede. </li></ol> 3. Preparação do Tecido <ol><li> Remova a parafina from o tecido por imersão em lâminas 2x frasco Coplin em xileno fresco durante 10 minutos cada e re-hidratar a secção de tecido por 5 min em incubações concentrações decrescentes de etanol (100%, 95% e 80%) em ddH 2 O. Execute esta etapa em um exaustor. </li><li> Mergulha-se o tecido em água tratada com DEPC, durante 5 min. Nesta altura, levar limites em torno secção de tecido com a pena barreira hidrofóbica. </li><li> Corrigir o tecido com PFA a 4% durante 15 min, seguido por uma lavagem com PBS. </li><li> Mergulha-se o tecido em 0,3% de Triton X-100 / PBS durante 10 minutos seguido por uma lavagem com PBS. </li><li> Incubar amostra com uma solução de / ul de proteinase K 50 ^ g durante 15 min a 37 ° C. Em seguida, incubar as secções de tecido com trietanolamina e anidrido acético durante 5 min à temperatura ambiente, seguido por uma lavagem com PBS. Sem lavagem com PBS é necessária entre o tratamento com proteinase K e / tratamento anidrido acético trietanolamina. </li></ol> 4. Hibridização <ol><li> Lavar o tecido completamente e incuBATE a secção de tecido com tampão de hibridação durante 2 a 3 horas à temperatura ambiente. Tipicamente 120 ul de tampão de hibridação é suficiente para cobrir a secção de tecido. </li><li> Diluir 5&#39;-DIG marcado com sonda LNA a concentração final de 3:00 em 120 ul de solução de hibridação. À medida que a concentração de estoque geralmente é de 40 ng / ul, adicionar 3 ul de estoque de tampão de hibridação de 120 ul e incubar mistura a 95 ° C durante 5 min. </li><li> Incubar secção de tecido com pernoite sonda a 54 ° C (Tm) em câmara úmida. Normalmente 120 mL é suficiente para cobrir uma seção. Criar umidade usando duas toalhas de papel molhado. O tamanho ideal da câmara é de 10 polegadas por 8 polegadas. </li><li> Nota: Tm pode ser diferente para diferentes miRNA. 54 ° C é de mencionar para miR-489. </li></ol> 5. lavagem rigorosa <ol><li> Lave o tecido com 5x SSC para 7 min (após o passo de hibridação, RNase condições livres não são mais necessários). </li><li> Lave a sagacidade secção de tecidoh 1x SSC durante 7 min, duas vezes a 57 ° C. </li><li> Lava-se a secção de tecido com SSC 0,2x durante 7 minutos a 57 ° C. </li><li> Lava-se a secção de tecido com SSC 0,2x durante 7 min à temperatura ambiente. </li><li> Incubar secção de tecido em PBS durante 10 min. </li></ol> 6. Bloqueio <ol><li> Incubar a secção de tecido com tampão de bloqueio à temperatura ambiente durante 1 hora numa câmara húmida (Para fazer um tampão de bloqueio e juntar 2 ml de FBS, 10 ul de Tween-20 e 3 pi de BSA em 18 m de PBS. Loja tampão de bloqueio a 4 ° C). </li></ol> 7. Anticorpo primário Incubação e Desenvolvimento <ol><li> Diluir 1: anticorpo anti-DIG-AP 300 em tampão de bloqueio e incubar a secção de tecido com o anticorpo, a 4 ° C durante a noite. </li><li> Lavar as seções com tampão de lavagem 3 vezes, durante 5 minutos cada (tampão fornecido pelo kit de desenvolvimento). </li><li> Desenvolver a secção de tecido com um kit comercial para fosfatase alcalina que produz CY-3 luz fluorescente. </li> &lt;li&gt; Lava-se a secção com tampão de lavagem durante 5 minutos. secção mancha com 100 ul de 2,5 ng / mL de corante Hoechst, durante 5 minutos, seguido de uma lavagem de 5 min com tampão de lavagem. </li><li> Montar o tampão com secção de montagem de acordo com o protocolo do fabricante. Use 15 ul por secção de tecido e limpe solução de montagem excessiva com lenço de papel. Selar o slide com unhas polonês claro para evitar vazamentos. </li><li> Observe o microscópio fluorescente seção usando com ampliação de 10x lente de modo total é de 100X. </li></ol>

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There is nothing to disclose.

O objetivo deste estudo foi determinar o nível de expressão miRNA em tecidos de câncer de mama utilizando miRNA hibridização in situ. Deve ser notado que nesta experiência, todas as condições foram optimizadas para o tecido do cancro da mama. Optimização adicional pode ser necessário para outros tipos de tecidos. Todas as soluções deve ser feita com água com DEPC e todos os recipientes a ser usado deve ser lavado com água tratada com DEPC e subsequentemente autoclavadas. Mesmo fraca contaminaçã...

O cancro da mama é um dos tumores malignos mais comuns que afetam a população feminina em todo o mundo. Mais de 1,3 milhões de casos de câncer de mama são notificados a cada ano no mundo 1,2. Embora as células tumorais têm sido tradicionalmente considerados como biologicamente homogênea e altamente proliferativa, tornou-se evidente que o câncer de mama é geneticamente e clinicamente heterogênea. A resistência aos fármacos anticancerígenos prevalentes é uma característica de cancros da mama avançado, conduzindo à mortalidade na maioria dos pacientes através da sua promoção da progressão do cancro e metástases à distância 3. MicroRNAs (miRNAs) são uma classe de pequenas moléculas de RNA de aproximadamente 22 nucleótidos de comprimento que regulam a expressão do gene através do bloqueio da tradução do mRNA ou mediar a degradação de ARNm 4. Mais de 2.000 miRNAs diferentes foram até agora identificados em humanos, que estão ligadas a doenças humanas 5. Uma crescente variedade de estudos have demonstraram que miARN pode funcionar como oncogenes e supressores tumorais e são frequentemente desreguladas em tumores 6. miARNs impacto fortemente toda a tumorigénese principal, controlando as principais reguladores da progressão do ciclo celular, senescência, apoptose e autofagia, juntamente com os da motilidade de células tumorais, invasão e metástase 7. Expressão aberrante miRNA foi também associada a implicações clínicas, tais como estágio, diferenciação, prognóstico e resposta ao adjuvante terapia 8. Em particular, o aumento da expressão de miR-146 está correlacionada com mau prognóstico em pacientes com câncer de pulmão 9. Outro estudo demonstrou que a expressão perdida de miRNA-let7b está ligada a fenótipos malignas e, consequentemente, pior sobrevida 6. Compreender os tipos de células e estruturas que expressam miRNAs é uma parte importante de compreender os mecanismos através dos quais alterações nas células de expressão influência miRNA etecido fenótipos 10. Certos miARNs encontrado para ser segregado em células do estroma são tomadas por células epiteliais e, especificamente, agir sobre os seus objectivos. Na mesma linha, o miR-212 e miR-132 que são segregadas por estroma e regular as interacções epiteliais-estromal requeridas para crescimento ductal durante 11 desenvolvimento da glândula mamária. O objetivo geral deste trabalho é explicar um protocolo detalhado para detectar a expressão de miRNA em tecidos de cancro da mama através de miRNA hibridização in situ. Temos todas as condições optimizadas para ensaiar os níveis de expressão de miARN-489 em amostras de pacientes de cancro da mama. Para este fim, foi utilizado um DIG marcado bloqueado ácido sonda (LNA) nucleico em nosso experimento. Alguns dos seus nucleótidos tinha uma modificação LNA, que é, uma ponte metileno que liga &#39;átomo de oxigénio e 4&#39; a 2 átomos de carbono do esqueleto ribose que fixa as sequências de nucleótidos de tal modo que ele fica &quot;bloqueado no lugar&quot; após a hibridização. Como resultado, é muito mais difícilpara o complexo hibridado para desnaturar 12,13.

<table><tbody><tr><td>ELF-97</td><td>Life technology&amp;nbsp;</td><td>E6604</td><td></td></tr><tr><td>50x Denhard&#039;t&amp;nbsp;</td><td>Life technology&amp;nbsp;</td><td>750018</td><td></td></tr><tr><td>t-RNA</td><td>Roche</td><td>109541</td><td>Reconstitute in DEPC water</td></tr><tr><td>Herring Sperm DNA</td><td>Promega&amp;nbsp;</td><td>D1815</td><td></td></tr><tr><td>Roche Blocking&amp;nbsp;</td><td>Roche</td><td>11096176001</td><td></td></tr><tr><td>RVC</td><td>Fisher</td><td>50-812-650</td><td>Before use spin down it at 16.1 RCF and take supernatant&amp;nbsp;</td></tr><tr><td>miR-489 probe&amp;nbsp;</td><td>Exiqon</td><td>38599-01</td><td></td></tr><tr><td>Nuclease free BSA</td><td>Roche&amp;nbsp;</td><td>711454</td><td></td></tr><tr><td>Primary antibody-anti DIG</td><td>Roche&amp;nbsp;</td><td>11093274910</td><td></td></tr><tr><td>Diethyl Pyrocarbonate&amp;nbsp;</td><td>Sigma</td><td>1609478</td><td></td></tr></tbody></table>

clinicopathological analysis of mirna expression in breast cancer tissues by using mirna in situ hybridization

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA (Locked Nucleic Acid) probe. The probe was recognized by anti-DIG alkaline phosphatase antibodies and later developed using alkaline phosphatase substrate producing fluorescence signals. Here we utilized miRNA in situ hybridization (MISH) technique to analyze expression of miR-489 in tissues from breast cancer patients. This technique can detect the localization of miRNA of interest in individual tissue samples. This technique can be used to compare the expression of desired miRNA in tumor tissue with that in adjacent normal tissue and to identify the specific structures responsible for expressing this miRNA. This technique can be very useful in answering certain clinical questions, such as role of specific miRNA in the development of cancer. Our results indicate that mammary epithelial cells express significantly higher levels of miR-489 than adjacent tumor cells.

tecido do cancro da mama humano foi utilizado para determinar a expressão de miARN-489. Ducto da glândula mamaria e células epiteliais foram encontrados para expressar significativamente mais elevados de miARN-489 níveis que o tecido adjacente do tumor em dois pacientes (Figura 1A e 1B). Isto demonstra claramente que a expressão de miARN-489 foi perdido no tecido tumoral, sugerindo que a actividade supressora do tumor de miARN-489. Para confirmar ai...

Watch this Scientific Journal Video about Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization at JoVE.com

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

Análise clínico-patológico de miRNA Expressão em cancro da mama tecidos usando miRNA<em&gt; In Situ</em&gt; Hibridização

In this article, we describe a detailed protocol for miRNA detection in breast cancer tissue using in situ hybridization with a digoxigenin-labelled LNA ...

clinicopathological-analysis-mirna-expression-breast-cancer-tissues

Research

JoVE Journal

Medicine

Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

6.7K visualizações.  University of South Carolina. Here we present a protocol to detect miRNA expression in breast cancer patient samples using miRNA in situ hybridization.

Vídeo: Clinicopathological Analysis of miRNA Expression in Breast Cancer Tissues by Using miRNA In Situ Hybridization

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

Sequenciação de MicroRNA retrospectiva: Biblioteca de DNA complementar preparação protocolo usando amostras de RNA de parafina fixada em formol

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Genética

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To date, miRNAs have been implicated in a large number of biological and disease processes, which has signified the need for the reliable detection methods of miRNA transcripts. Here, we describe a detailed protocol for digoxigenin-labeled (DIG) Locked Nucleic Acid (LNA) probe-based miRNA detection, combined with protein immunostaining on mouse heart sections. First, we performed an in situ hybridization technique using the probe to identify miRNA-182 expression in heart sections from control and cardiac hypertrophy mice. Next, we performed immunostaining for cardiac Troponin T (cTnT) protein, on the same sections, to co-localize miRNA-182 with the cardiomyocyte cells. Using this protocol, we were able to detect miRNA-182 through an alkaline phosphatase based colorimetric assay, and cTnT through fluorescent staining. This protocol can be used to detect the expression of any miRNA of interest through DIG-labeled LNA probes, and relevant protein expression on mouse heart tissue sections.

Tissue-specific miRNA Expression Profiling in Mouse Heart Sections Using In Situ Hybridization

micro-RNAs (miRNAs) are short and highly homologous RNA sequences, serving as post-transcriptional regulators of messenger RNAs (mRNAs). Current miRNA detection methods vary in sensitivity and specificity. We describe a protocol that combines in situ hybridization and immunostaining for concurrent detection of miRNA and protein molecules on mouse heart tissue sections.

Tecido-específica miRNA Expression Profiling Mouse coração seções usando em hibridação in Situ

micro-RNAs (miRNAs) are single-stranded RNA transcripts that bind to messenger RNAs (mRNAs) and inhibit their translation or promote their degradation. To ...

Detection of a Circulating MicroRNA Custom Panel in Patients with Metastatic Colorectal Cancer

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful biomarkers for clinical practice. Here, we present a protocol to extract, reverse transcribe and evaluate the expression levels of circulating miRNAs from plasma samples of patients with colorectal cancer (CRC). microRNAs (miRNAs) are a class of non-coding RNAs of 18-25 nucleotides in length that regulate the expression of target genes at the translational level and play an important role, including that of pro- and anti-angiogenic function, in the physiopathology of various organs. miRNAs are stable in biological fluids such as serum and plasma, which renders them ideal circulating biomarkers for cancer diagnosis, prognosis and treatment decision-making and monitoring. Circulating miRNA extraction was performed using a rapid and effective method that involves both organic and column-based methodologies. For miRNA retrotranscription, we used a multi-step procedure that considers polyadenylation at 3&#39; and the ligation of an adapter at 5&#39; of the mature miRNAs, followed by random miRNA pre-amplification. We selected a 24-miRNA custom panel to be tested by quantitative real-time polymerase chain reaction (qRT-PCR) and spotted the miRNA probes on array custom plates. We performed qRT-PCR plate runs on a real-time PCR System. Housekeeping miRNAs for normalization were selected using GeNorm software (v. 3.2). Data were analyzed using Expression suite software (v 1.1) and statistical analyses were performed. The method proved to be reliable and technically robust and could be useful to evaluate biomarker levels in liquid samples such as plasma and/or serum.

We present a protocol to evaluate the expression levels of circulating microRNA (miRNAs) in plasma samples from cancer patients. In particular, we used a commercially available kit for circulating miRNA extraction and reverse transcription. Finally, we analyzed a panel of 24 selected miRNAs using real-time pre-spotted probe custom plates.

Deteção de um painel personalizado de MicroRNA circulantes em pacientes com câncer colorretal metastático

There is increasing interest in liquid biopsy for cancer diagnosis, prognosis and therapeutic monitoring, creating the need for reliable and useful ...

Pesquisa do câncer

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

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

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

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

Detecção MicroRNA em tumores de próstata por Quantitative PCR em tempo real (qPCR)

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

Medicina

Analysis of Combinatorial miRNA Treatments to Regulate Cell Cycle and Angiogenesis

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is unregulated proliferation and cell division. Inhibition of proliferation by halting cell cycle progression has been shown to be a promising approach for cancer treatment, including LC. 
miRNA therapeutics have emerged as important post-transcriptional gene regulators and are increasingly being studied for use in cancer treatment. In recent work, we utilized two miRNAs, miR-143 and miR-506, to regulate cell cycle progression. A549 non-small cell lung cancer (NSCLC) cells were transfected, gene expression alterations were analyzed, and apoptotic activity due to the treatment was finally analyzed. Downregulation of cyclin-dependent kinases (CDKs) were detected (i.e., CDK1, CDK4 and CDK6), and cell cycle halted at the G1/S and G2/M phase transitions. Pathway analysis indicated potential antiangiogenic activity of the treatment, which endows the approach with multifaceted activity. Here, described are the methodologies used to identify miRNA activity regarding cell cycle inhibition, induction of apoptosis, and effects of treatment on endothelial cells by inhibition of angiogenesis. It is hoped that the methods presented here will support future research on miRNA therapeutics and corresponding activity and that the representative data will guide other researchers during experimental analyses.

miRNA therapeutics have significant potential in regulating cancer progression. Demonstrated here are analytical approaches used for identification of the activity of a combinatorial miRNA treatment in halting cell cycle and angiogenesis.

Análise de combinatória miRNA tratamentos para regular o ciclo de celular e a angiogênese

Lung cancer (LC) is the leading cause of cancer-related deaths worldwide. Similar to other cancer cells, a fundamental characteristic of LC cells is ...

An In Vitro Protocol for Evaluating MicroRNA Levels, Functions, and Associated Target Genes in Tumor Cells

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including cancers. These small regulatory RNAs mainly interact with the 3&#8217; untranslated regions (3&#8217; UTR) of their target messenger RNAs (mRNAs) ultimately resulting in the inhibition of decoding processes of mRNAs and the augmentation of target mRNA degradations. Based on the expression levels and intracellular functions, miRNAs are able to serve as regulatory factors of oncogenic and tumor-suppressive mRNAs. Identification of bona fide target genes of a miRNA among hundreds or even thousands of computationally predicted targets is a crucial step to discern the roles and basic molecular mechanisms of a miRNA of interest. Various miRNA target prediction programs are available to search possible miRNA-mRNA interactions. However, the most challenging question is how to validate direct target genes of a miRNA of interest. This protocol describes a reproducible strategy of key methods on how to identify miRNA targets related to the function of a miRNA. This protocol presents a practical guide on step-by-step procedures to uncover miRNA levels, functions, and related target mRNAs using the probe-based real-time polymerase chain reaction (PCR), sulforhodamine B (SRB) assay following a miRNA mimic transfection, dose-response curve generation, and luciferase assay along with the cloning of 3&#8217; UTR of a gene, which is necessary for proper understanding of the roles of individual miRNAs.

This protocol uses a probe-based real-time polymerase chain reaction (PCR), a sulforhodamine B (SRB) assay, 3&#8217; untranslated regions (3&#8217; UTR) cloning, and a luciferase assay to verify the target genes of a miRNA of interest and to understand the functions of miRNAs.

Protocolo in vitro para avaliação de níveis de MicroRNA, funções e genes alvo associados em células tumorais

MicroRNAs (miRNAs) are small regulatory RNAs which are recognized to modulate numerous intracellular signaling pathways in several diseases including ...

Profiling of Estrogen-regulated MicroRNAs in Breast Cancer Cells

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen receptors (ERs), ER&#945;, and ER&#946;. ER&#945; is frequently upregulated in breast cancer and drives the proliferation of breast cancer cells. The ERs function as transcription factors and regulate gene expression. Whereas ER&#945;&#39;s regulation of protein-coding genes is well established, its regulation of noncoding microRNA (miRNA) is less explored. miRNAs play a major role in the post-transcriptional regulation of genes, inhibiting their translation or degrading their mRNA. miRNAs can function as oncogenes or tumor suppressors&#160;and are also promising biomarkers. Among the miRNA assays available, microarray and quantitative real-time polymerase chain reaction (qPCR) have been extensively used to detect and quantify miRNA levels. To identify miRNAs regulated by estrogen signaling in breast cancer, their expression in ER&#945;-positive breast cancer cell lines were compared before and after estrogen-activation using both the &#181;Paraflo-microfluidic microarrays and Dual Labeled Probes-low density arrays. Results were validated using specific qPCR assays, applying both Cyanine dye-based and Dual Labeled Probes-based chemistry. Furthermore, a time-point assay was used to identify regulations over time. Advantages of the miRNA assay approach used in this study is that it enables a fast screening of mature miRNA regulations in numerous samples, even with limited sample amounts. The layout, including the specific conditions for cell culture and estrogen treatment, biological and technical replicates, and large-scale screening followed by in-depth confirmations using separate techniques, ensures a robust detection of miRNA regulations,&#160;and eliminates false positives and other artifacts. However, mutated or unknown miRNAs, or regulations at the primary and precursor transcript level, will not be detected. The method presented here represents a thorough investigation of estrogen-mediated miRNA regulation.

Molecular signaling through both estrogen and microRNAs are critical in breast cancer development and growth. Estrogen activates the estrogen receptors, which are transcription factors. Many transcription factors can regulate the expression of microRNAs, and estrogen-regulated microRNAs can be profiled using different large-scale techniques.

Profiling de MicroRNAs estrogênio regulados em células do cancro da mama

Estrogen plays vital roles in mammary gland development and breast cancer progression. It mediates its function by binding to and activating the estrogen ...

Optimization of a Multiplex RNA-based Expression Assay Using Breast Cancer Archival Material

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. Gene amplification and expression diagnostic testing using archival material or material that requires transportation to servicing laboratories, needs a more robust and accurate test adapted to current clinical workflows. Our research team optimized the use of Invitrogen&#8482; QuantiGene&#8482; Plex Assay (Thermo Fisher Scientific) to quantify RNA in archival material using branched-DNA (bDNA) technology on Luminex xMAP&#174; magnetic beads. The gene expression assay described in this manuscript is a novel, quick, and multiplex method that can accurately classify breast cancer into the different molecular subtypes, omitting the subjectivity of interpretation inherent in imaging techniques. In addition, due to the low input of material required, heterogeneous tumors can be laser microdissected using Hematoxylin and Eosin (H&#38;E) stained sections. This method has a wide range of possible applications including tumor classification with diagnostic potential and measurement of biomarkers in liquid biopsies, which would allow better patient management and disease monitoring. In addition, the quantitative measurement of biomarkers in archival material is useful in oncology research with access to libraries of clinically-annotated material, in which retrospective studies can validate potential biomarkers and their clinical outcome correlation.

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic services in pathology laboratories worldwide. This manuscript describes the optimization of a panel of biomarkers using a multiplex magnetic bead assay to classify breast cancers.

Otimização de um ensaio de expressão baseada em RNA Multiplex usando Material de arquivo de câncer de mama

Nucleic acid degradation in archival tissue, tumor heterogeneity, and a lack of fresh frozen tissue specimens can negatively impact cancer diagnostic ...

Bioengenharia

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, diagnosis, prognosis and treatment. In recent years, microRNAs (miRNAs) have emerged as promising alternate biomarkers for prostate cancer diagnosis and prognosis. However, the development of miRNAs as effective biomarkers for prostate cancer heavily relies on their accurate detection in clinical tissues. miRNA analyses in prostate cancer clinical specimens is often challenging owing to tumor heterogeneity, sampling errors, stromal contamination etc. The goal of this article is to describe a simplified workflow for miRNA analyses in archived FFPE or fresh frozen prostate cancer clinical specimens using a combination of quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH). Within this workflow, we optimize the existing methodologies for miRNA extraction from FFPE and frozen prostate tissues and expression analyses by Taqman-probe based miRNA RT-PCR. In addition, we describe an optimized method for ISH analyses formiRNA detection in prostate tissues using locked nucleic acid (LNA)- based probes. Our optimized miRNA ISH protocol can be applied to prostate cancer tissue slides or prostate cancer tissue microarrays (TMA).

Here we describe a simplified protocol for microRNA (miRNA) expression analyses in archived Formalin-Fixed, Paraffin-Embedded (FFPE) or fresh frozen prostate cancer (PCa) clinical tissues employing quantitative real-time PCR (RT-PCR) and in situ hybridization (ISH).

As análises de expressão de miRNA no câncer de próstata tecidos clínicos

A critical challenge in prostate cancer (PCa) clinical management is posed by the inadequacy of currently used biomarkers for disease screening, ...

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA probes. This protocol, originally developed by Kloosterman and colleagues for broad use with Exiqon miRNA probes1, has been modified to overcome challenges inherent in miRNA analysis in kidney tissues. These include issues such as structure identification and hard to remove residual probe and antibody. Use of relatively thin, 5 mm thick, tissue sections allowed for clear visualization of kidney structures, while a strong probe signal was retained in cells. Additionally, probe concentration and incubation conditions were optimized to facilitate visualization of microRNA expression with low background and nonspecific signal. Here, the optimized protocol is described, covering the initial tissue collection and preparation through the mounting of slides at the end of the procedure. The basic components of this protocol can be altered for application to other tissues and cell culture models.

MicroRNA In situ Hybridization for Formalin Fixed Kidney Tissues

This article describes an in situ hybridization protocol optimized for colormetric detection of microRNA expression in formalin fixed kidney sections.

MicroRNA<em&gt; Em situ</em&gt; Hibridização de tecidos de rim fixadas em formalina

In this article we describe a method for colorimetric detection of miRNA in the kidney through in situ hybridization with digoxigenin tagged microRNA ...

Análise clínico-patológico de miRNA Expressão em cancro da mama tecidos usando miRNA

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