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.

Questo studio è stato approvato dal Institutional Review Board dell&#39;Università Hwasun Hospital Chonnam Nazionale e University of South Carolina. campioni TMA composte da cancro al seno e tessuti del seno normali adiacenti abbinati sono stati forniti dalla Biobanca di Chonnam National University Hospital Hwasun, membro della Corea del Biobanca rete. 1. Soluzione Preparazione <ol><li> Preparare 1 L di DNasi e RNasi acqua libera. Effettuare tutte le soluzioni chimiche che utilizzano dietilpirocarbonato (DEPC) acqua trattata. Trattare 1 L di acqua con 500 ml di DEPC e incubare per 3 ore a temperatura ambiente. Autoclave l&#39;acqua per inattivare DEPC. Attenzione: Non inalare DEPC poiché è noto agente cancerogeno. </li><li> Preparare 1x PBS da 10x PBS con acqua DEPC trattati. </li><li> Preparare 20x SSC (sciogliere 175,3 g di NaCl e 88,2 g di citrato di sodio in 800 ml di acqua, regolare il pH a 7,0 con poche goccia di 14 N soluzione di HCl e regolare il volume di 1 L con water), 4% paraformaldeide, 95% di etanolo, 80% di etanolo con acqua DEPC trattati. </li><li> Aggiungere 8 ml di trietanolamina e 1,05 ml di HCl in 590 ml di acqua trattata con DEPC e aggiungere 1,5 ml di anidride acetica per acetilazione. </li><li> Preparare 50 mg / mL proteinasi K in tampone (50 mM Tris-HCl, pH 8,0 in DEPC acqua trattata). </li><li> Preparare tampone di ibridazione composta da 500 ml di formammide, 250 microlitri di 20x SSC, 100 ml di 50x soluzione di Denhardt, 12,5 l di t-RNA, 2,5 l di DNA aringhe sperma, 30 ml di RVC, 0,02 g di blocco dei reagenti e 50 ml di acqua DEPC. Preparare fresco. </li></ol> 2. tessuto mammario Sezioni <ol><li> Tagliare 5 micron sezioni incluse in paraffina fissati in formalina con microtomo e applicare la sezione per caricare slitta 14. Nota: il tessuto del seno utilizzato qui è stato preparato in precedenza dalla Corea del Biobanche di rete. </li></ol> 3. Preparazione del tessuto <ol><li> Rimuovere la paraffina from tessuto immergendo i vetrini in Coplin 2x vaso in xilene fresco per 10 minuti ciascuno e reidratare la sezione di tessuto da 5 min incubazioni in concentrazioni decrescenti di etanolo (100%, 95% e 80%) in DDH 2 O. Eseguire questo passaggio in una cappa aspirante. </li><li> Immergere il tessuto in DEPC acqua trattata per 5 min. A questo punto fare confini intorno sezione di tessuto con la penna barriera idrofoba. </li><li> Fissare il tessuto con 4% PFA per 15 minuti seguita da un lavaggio con PBS. </li><li> Immergere il tessuto in 0,3% Triton X-100 / PBS per 10 minuti seguita da un lavaggio con PBS. </li><li> Incubare campione con 50 mg soluzione / ml proteinasi K per 15 minuti a 37 ° C. Poi incubare sezioni di tessuto con trietanolammina e anidride acetica per 5 min a temperatura ambiente, seguito da un lavaggio con PBS. Nessun lavaggio PBS è richiesto tra il trattamento proteinasi K e trietanolammina / trattamento anidride acetica. </li></ol> 4. ibridazione <ol><li> Lavare accuratamente e incu il tessutobate la sezione di tessuto con tampone di ibridazione da 2 a 3 ore a temperatura ambiente. Tipicamente 120 microlitri tampone di ibridazione è sufficiente a coprire la sezione di tessuto. </li><li> Diluire 5&#39;-DIG etichettata sonda LNA a concentrazione finale di 3:00 in 120 ml soluzione di ibridazione. Come la concentrazione stock di solito è di 40 ng / ml, aggiungere 3 ml di brodo di tampone di ibridazione 120 ml e incubare la miscela a 95 ° C per 5 min. </li><li> Incubare la sezione di tessuto con sonda durante la notte a 54 ° C (Tm) in una camera umida. Tipicamente 120 ml è sufficiente a coprire una sezione. Creare umidità utilizzando due asciugamani di carta bagnati. La dimensione ideale della camera è di 10 pollici da 8 pollici. </li><li> Nota: Tm può essere diverso per i diversi miRNA. 54 ° C è menzione per miR-489. </li></ol> 5. stringenza Wash <ol><li> Lavare il tessuto con 5x SSC per 7 minuti (dopo la fase di ibridazione, RNasi condizioni di libero non sono più necessari). </li><li> Lavare l&#39;arguzia sezione di tessutoh 1x SSC per 7 minuti due volte a 57 ° C. </li><li> Lavare la sezione di tessuto con 0.2x SSC per 7 minuti a 57 ° C. </li><li> Lavare la sezione di tessuto con 0.2x SSC per 7 minuti a temperatura ambiente. </li><li> Incubare sezione di tessuto in PBS per 10 min. </li></ol> 6. Blocco <ol><li> Incubare la sezione di tessuto con tampone di arresto a temperatura ambiente per 1 ora in camera umida (Per fare un tampone di bloccaggio con 2 ml di FBS, 10 ml di Tween-20 e 3 ml di BSA in 18 m di PBS. Conservare tampone di bloccaggio a 4 ° C). </li></ol> 7. incubazione dell&#39;anticorpo primario e lo Sviluppo <ol><li> Diluire 1: 300 di anticorpi anti-DIG-AP in tampone di bloccaggio e incubare la sezione di tessuto con l&#39;anticorpo a 4 ° C durante la notte. </li><li> Lavare le sezioni con tampone di lavaggio per 3 volte, per 5 minuti ciascuna (buffer fornito dal kit di sviluppo). </li><li> Sviluppare la sezione di tessuto con un kit commerciale per la fosfatasi alcalina che produce CY-3 luce fluorescente. </li> &lt;li&gt; Lavare la sezione con tampone di lavaggio per 5 minuti. sezione macchia con 100 ml di 2,5 ng / ml colorante Hoechst per 5 minuti seguita da 5 minuti di lavaggio con tampone di lavaggio. </li><li> Montare la sezione con il tampone di montaggio secondo il protocollo del produttore. Utilizzare 15 ml per sezione di tessuto e pulire soluzione di montaggio eccessiva con il tessuto pulire. Sigillare il vetrino con smalto trasparente per evitare perdite. </li><li> Osservare il microscopio sezione fluorescente utilizzando con ingrandimento 10X lente in modo totale è di 100X. </li></ol>

<ol>
	<li>Shah, N. R., Chen, H. <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+pathogenesis+of+breast+cancer:+Implications+in+diagnosis+and+treatment.">MicroRNAs in pathogenesis of breast cancer: Implications in diagnosis and treatment.</a> World J Clin Oncol. 5, 48-60 (2014).</li><li>Tang, H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-185+suppresses+tumor+proliferation+by+directly+targeting+E2F6+and+DNMT1+and+indirectly+upregulating+BRCA1+in+triple-negative+breast+cancer.">miR-185 suppresses tumor proliferation by directly targeting E2F6 and DNMT1 and indirectly upregulating BRCA1 in triple-negative breast cancer.</a> Mol Cancer Ther. 13, 3185-3197 (2014).</li><li>Ye, X. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Epigenetic+silencing+of+miR-375+induces+trastuzumab+resistance+in+HER2-positive+breast+cancer+by+targeting+IGF1R.">Epigenetic silencing of miR-375 induces trastuzumab resistance in HER2-positive breast cancer by targeting IGF1R.</a> BMC Cancer. 14, 134 (2014).</li><li>Oneyama, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-mediated+downregulation+of+mTOR/FGFR3+controls+tumor+growth+induced+by+Src-related+oncogenic+pathways.">MicroRNA-mediated downregulation of mTOR/FGFR3 controls tumor growth induced by Src-related oncogenic pathways.</a> Oncogene. 30, 3489-3501 (2011).</li><li>McGuire, A., Brown, J. A., Kerin, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Metastatic+breast+cancer:+the+potential+of+miRNA+for+diagnosis+and+treatment+monitoring.">Metastatic breast cancer: the potential of miRNA for diagnosis and treatment monitoring.</a> Cancer Metastasis Rev. 34, 145-155 (2015).</li><li>Quesne, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+and+prognostic+associations+of+miR-205+and+let-7b+in+breast+cancer+revealed+by+in+situ+hybridization+analysis+of+micro-RNA+expression+in+arrays+of+archival+tumour+tissue.">Biological and prognostic associations of miR-205 and let-7b in breast cancer revealed by in situ hybridization analysis of micro-RNA expression in arrays of archival tumour tissue.</a> J Pathol. 227, 306-314 (2012).</li><li>Fernandez, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-340+inhibits+tumor+cell+proliferation+and+induces+apoptosis+by+targeting+multiple+negative+regulators+of+p27+in+non-small+cell+lung+cancer.">miR-340 inhibits tumor cell proliferation and induces apoptosis by targeting multiple negative regulators of p27 in non-small cell lung cancer.</a> Oncogene. 34, 3240-3250 (2015).</li><li>Yu, L., Zhang, J., Guo, X., Li, Z., Zhang, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA-224+upregulation+and+AKT+activation+synergistically+predict+poor+prognosis+in+patients+with+hepatocellular+carcinoma.">MicroRNA-224 upregulation and AKT activation synergistically predict poor prognosis in patients with hepatocellular carcinoma.</a> Cancer Epidemiol. 38, 408-413 (2014).</li><li>Li, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=microRNA-146+up-regulation+predicts+the+prognosis+of+non-small+cell+lung+cancer+by+miRNA+in+situ+hybridization.">microRNA-146 up-regulation predicts the prognosis of non-small cell lung cancer by miRNA in situ hybridization.</a> Exp Mol Pathol. 96, 195-199 (2014).</li><li>Kriegel, A. J., Liang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+in+situ+hybridization+for+formalin+fixed+kidney+tissues.">MicroRNA in situ hybridization for formalin fixed kidney tissues.</a> J Vis Exp. , e50785 (2013).</li><li>Ucar, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-212+and+miR-132+are+required+for+epithelial+stromal+interactions+necessary+for+mouse+mammary+gland+development.">miR-212 and miR-132 are required for epithelial stromal interactions necessary for mouse mammary gland development.</a> Nat Genet. 42, 1101-1108 (2010).</li><li>Nuovo, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+microRNAs+in+paraffin+embedded,+formalin+fixed+tissues+and+the+co-localization+of+their+putative+targets.">In situ detection of microRNAs in paraffin embedded, formalin fixed tissues and the co-localization of their putative targets.</a> Methods. 52, 307-315 (2010).</li><li>Kierzek, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+influence+of+locked+nucleic+acid+residues+on+the+thermodynamic+properties+of+2'-O-methyl+RNA/RNA+heteroduplexes.">The influence of locked nucleic acid residues on the thermodynamic properties of 2'-O-methyl RNA/RNA heteroduplexes.</a> Nucleic Acids Res. 33, 5082-5093 (2005).</li><li>Fischer, A. H., Jacobson, K. A., Rose, J., Zeller, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Paraffin+embedding+tissue+samples+for+sectioning.">Paraffin embedding tissue samples for sectioning.</a> CSH Protoc. 2008, (2008).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sequential+expression+of+miR-182+and+miR-503+cooperatively+targets+FBXW7,+contributing+to+the+malignant+transformation+of+colon+adenoma+to+adenocarcinoma.">Sequential expression of miR-182 and miR-503 cooperatively targets FBXW7, contributing to the malignant transformation of colon adenoma to adenocarcinoma.</a> J Pathol. 234, 488-501 (2014).</li></ol>

There is nothing to disclose.

L&#39;obiettivo di questo studio era di determinare il livello di espressione dei miRNA nei tessuti di cancro al seno utilizzando miRNA ibridazione in situ. Si deve notare che in questo esperimento, tutte le condizioni sono stati ottimizzati per il tessuto del cancro al seno. Ulteriore ottimizzazione potrebbe essere richiesto per altri tipi di tessuto. Tutte le soluzioni devono essere effettuate con acqua DEPC e tutti i contenitori da utilizzare devono essere risciacquati con acqua DEPC trattati e successivamen...

Il cancro al seno è uno dei tumori più comuni che colpiscono la popolazione femminile in tutto il mondo. Oltre 1,3 milioni di casi di cancro al seno sono segnalati ogni anno in tutto il mondo 1,2. Anche se le cellule tumorali sono stati tradizionalmente considerati come biologicamente omogenea e altamente proliferative, è diventato evidente che il cancro al seno è geneticamente e clinicamente eterogenea. La resistenza ai farmaci antitumorali prevalenti è una caratteristica dei tumori al seno avanzato, che porta alla mortalità nella maggior parte dei pazienti attraverso la sua facilitazione della progressione del cancro e le metastasi a distanza 3. I microRNA (miRNA) sono una classe di piccole molecole di RNA lunghi circa 22 nucleotidi che regolano l&#39;espressione genica bloccando traduzione dell&#39;mRNA o mediare mRNA degrado 4. Più di 2.000 differenti miRNA sono stati finora identificati nell&#39;uomo, che sono collegati a malattie umane 5. Una crescente serie di studi have ha dimostrato che miRNA possono funzionare come oncogeni e soppressori tumorali e sono spesso disregolazione nei tumori 6. miRNA impatto fortemente tutta la tumorigenesi primaria controllando i principali regolatori del ciclo cellulare, la senescenza, l&#39;apoptosi e autofagia, insieme a quelli della motilità delle cellule tumorali, invasione e metastasi 7. Espressione aberrante miRNA è stata anche associata con implicazioni cliniche, come fase, la differenziazione, la prognosi e la risposta alla terapia adiuvante 8. In particolare, l&#39;aumento di espressione di miR-146 è correlato con una scarsa prognosi nei pazienti affetti da cancro del polmone 9. Un altro studio ha dimostrato che l&#39;espressione perduto di miRNA-let7b è legata a fenotipi maligni e, di conseguenza, scarsa sopravvivenza 6. Capire i tipi di cellule e strutture che esprimono miRNA è una parte importante di comprensione dei meccanismi attraverso i quali alterazioni delle cellule miRNA espressione e influenzatessuto fenotipi 10. Alcuni miRNA trovati ad essere secreto nelle cellule stromali sono prese in dalle cellule epiteliali e in particolare agiscono sui loro obiettivi. Allo stesso modo, miR-212 e miR-132 sono secreti dalle stroma e regolano le interazioni epiteliali-stromale necessari per l&#39;escrescenza duttale durante lo sviluppo della ghiandola mammaria 11. L&#39;obiettivo generale di questo articolo è quello di spiegare un protocollo dettagliato per rilevare miRNA nei tessuti di cancro al seno attraverso miRNA ibridazione in situ. Abbiamo ottimizzato tutte le condizioni per saggiare i livelli di espressione miRNA-489 nei campioni dei pazienti di cancro al seno. A tal fine, abbiamo utilizzato uno scavo con l&#39;etichetta bloccato sonda di acido nucleico (LNA) nel nostro esperimento. Alcuni dei suoi nucleotidi avevano una modifica LNA, cioè, un ponte che collega metilene &#39;atomo di ossigeno e 4&#39; del 2 atomi di carbonio dello scheletro ribosio che fissa il nucleotide tale che rimanga &quot;blocchi&quot; dopo ibridazione. Come risultato, è molto più difficileper il complesso ibridizzato denaturare 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.

tessuto del cancro al seno umano è stato utilizzato per determinare l&#39;espressione miRNA-489. Mammaria condotto ghiandola e cellule epiteliali sono stati trovati per esprimere significativamente più elevati miRNA-489 livelli di tessuto adiacente tumorale in due pazienti (Figura 1A e 1B). Ciò dimostra chiaramente che l&#39;espressione miRNA-489 è stato perso nel tessuto tumorale, suggerendo tumore attività soppressiva di miRNA-489. Ad ulteriore co...

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.

Analisi clinicopatologica di miRNA espressione in Breast Cancer tessuti mediante l&#39;utilizzo di miRNA<em&gt; In Situ</em&gt; ibridazione

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

Universidad de Cartagena UDEC

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Medicine

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

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

Video: 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.

Sequenziamento di MicroRNA retrospettiva: Protocollo di preparazione libreria DNA complementare utilizzando campioni di RNA di paraffina-incastonato formalina-fisso

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

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Genetica

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.

Tessuto-specifici miRNA Expression Profiling in Mouse cuore sezioni mediante ibridazione 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.

Rilevamento di un pannello personalizzato di MicroRNA circolanti in pazienti con cancro colorettale metastatico

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

Ricerca sul cancro

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

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

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

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

Rilevamento MicroRNA in tumori della prostata dal Quantitative Real-time PCR (qPCR)

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

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.

Analisi di miRNA combinatoria trattamenti per regolare il ciclo cellulare e l'angiogenesi

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.

Un protocollo in vitro per la valutazione dei livelli, delle funzioni e dei geni target associati nelle cellule tumorali

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 di microRNA regolati dagli estrogeni in cellule di carcinoma mammario

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.

Ottimizzazione di un'analisi di espressione di RNA-ha basato multisala usando materiale archivistico di cancro al seno

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

Bioingegneria

miRNA Expression Analyses in Prostate Cancer Clinical Tissues

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

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

Analisi miRNA nel tumore alla prostata tessuti cliniche

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

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; In situ</em&gt; Ibridazione per tessuti renali fissati in formalina

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

Analisi clinicopatologica di miRNA espressione in Breast Cancer tessuti mediante l'utilizzo di miRNA

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