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.

Bu çalışma Kurumsal Gözden Chonnam Ulusal Üniversitesi Hwasun Hastanesi Yönetim Kurulu ve Güney Carolina Üniversitesi tarafından onaylanmıştır. Meme kanseri ve eşleşen bitişik normal meme doku oluşan TMA örnekleri Chonnam Ulusal Üniversitesi Hwasun Hastanesi, Kore Biobank Ağının bir üye Biobank tarafından temin edildi. 1. Solüsyon Hazırlama <ol><li> DNaz ve RNaz içermeyen su 1 L hazırlayın. diethylpyrocarbonate (DEPC) arıtılmış su kullanan tüm kimyasal çözümler olun. DEPC 500 ul su ile 1 L tedavi ve oda sıcaklığında 3 saat inkübe edilir. DEPC inaktive su Otoklav. Dikkat: karsinojen bilindiğinden DEPC teneffüs etmeyin. </li><li> DEPC arıtılmış su kullanılarak 10x PBS 1x PBS hazırlayın. </li><li> 20x SSC (NaCl 175.3 g ve 800 ml su içinde sodyum sitrat 88.2 g çözülür hazırlanması HCI 14 N çözelti bir kaç damla 7.0 pH ayarlamak ve wat 1 L ses seviyesini ayarlamakER),% 4 paraformaldehit,% 95 etanol,% 80 etanol DEPC işlenmiş su ile. </li><li> 8 trietanolamin ilave edildi ve DEPC ile muamele edilmiş su 590 ml HCI 1.05 ml ilave edilir ve asetilasyonu için asetik anhidrid 1.5 ml ilave ediniz. </li><li> tampon maddesi içinde 50 ug / ml proteinaz K hazırlama (50 mM Tris-HCI, DEPC pH 8.0 su muamele edilmiş). </li><li> formamid 500 ul 20x SSC 250 ul, 50x Denhardt çözeltisi, 100 uL, t-RNA 12.5 ul, ringa sperm DNA&#39;sı 2.5 ul, RVC 30 ul, bloke reaktif ve 50 ul 0.02 g oluşan hibridizasyon tamponu hazırlayın DEPC su. taze hazırlayın. </li></ol> 2. Meme Doku Bölümler <ol><li> Mikrotom ile 5 mikron formalin ile fikse parafine gömülü bölümleri kesmek ve slayt 14 şarj etmek bölüm uygulayın. Not: Burada kullanılan meme dokusu Kore Biobank Network önceden hazırlanmıştır. </li></ol> 3. Doku Hazırlanması <ol><li> fro parafin kaldır10 dakika boyunca, taze ksilen Coplin kavanoza 2x her bir slayt daldırarak doku m ve GKD 2 O etanol (% 100,% 95 ve% 80) ve azalan konsantrasyonlarda 5 dakika inkubasyon doku bölümü rehidrasyonu Davlumbaz bu adımı gerçekleştirin. </li><li> DEPC doku 5 dakika arıtılmış su bırakın. Bu noktada hidrofobik bariyer kalemle doku bölümünde etrafında sınırları yapmak. </li><li> PBS ile yıkama, ardından 15 dakika boyunca% 4 PFA doku çözmek. </li><li> PBS ile yıkama, ardından 10 dakika boyunca% 0.3 Triton X-100 / PBS doku bırakın. </li><li> 37 ° C&#39;de 15 dakika boyunca 50 ug / ml Proteinaz K çözeltisi ile örnek inkübe edin. Daha sonra PBS ile bir yıkama ve ardından, oda sıcaklığında 5 dakika için, trietanolamin ve asetik anhidrid ile doku bölümleri inkübe edin. Hiçbir PBS yıkama Proteinaz K tedavisi ve trietanolamin / asetik anhidrit tedavisi arasında gereklidir. </li></ol> 4. Melezleme <ol><li> İyice ve incu doku yıkayınOda sıcaklığında 2 ila 3 saat için hibritleme tampon maddesi ile asitleme doku kesiti. Tipik 120 ul hibridizasyon tamponu doku bölümünü karşılamak için yeterli olduğunu. </li><li> Seyreltik 5&#39;-DIG 120 ul hibridizasyon çözeltisi içinde 3 uM nihai konsantrasyona kadar LNA etiketlenmiş prob. stok konsantrasyonu genellikle 40 ng / | il olduğu için, 120 ul hibridizasyon tamponu stok 3 ul ilave et ve 5 dakika boyunca 95 ° C&#39;de karışım inkübe edin. </li><li> nemli bir oda içinde 54 ° C&#39;de (Tm) prob gece boyunca doku bölümü inkübe edin. Tipik 120 ul bir bölüm karşılamak için yeterlidir. İki ıslak kağıt havlu kullanılarak nem oluşturun. odasının ideal boyutu 8 inç 10 inç. </li><li> Not: Tm Farklı miRNA için farklı olabilir. 54 ° C miR-489 için söz olduğunu. </li></ol> 5. sıkılık yıkama <ol><li> 7 dakika boyunca 5x SSC ile doku yıkayın (melezleme aşamasından sonra, RNaz ücretsiz koşullar artık gerekli vardır). </li><li> doku kesiti wit yıkayın57 ° C &#39;de iki kez 7 dakika süre ile H, 1x SSC. </li><li> 57 ° C &#39;de, 7 dakika için 0,2 X SSC ile doku kesiti yıkayın. </li><li> Oda sıcaklığında 7 dakika boyunca 0,2 X SSC ile doku kesiti yıkayın. </li><li> 10 dakika boyunca PBS içinde doku kesiti inkübe edin. </li></ol> 6. Engelleme <ol><li> (Tampon PBS 18 m 2 mi FBS, Tween-20, 10 ul BSA 3 ul bloke yapmak için. Mağazası 4 ° C&#39;de tampon bloke nemli bir oda içinde, 1 saat boyunca oda sıcaklığında bloke edici tampon ile doku kesiti inkübe ° C). </li></ol> 7. Birincil Antikor Kuluçka ve Kalkınma <ol><li> bloke edici tampon içinde 300, anti-DIP-AP antikor ve gece boyunca 4 ° C &#39;de antikor ile doku kesiti inkübe: 1 seyreltin. </li><li> Her biri 5 dakika (geliştirme kiti tarafından sağlanan tampon maddesi) için, yıkama tamponu ile 3 kez bölümleri yıkayın. </li><li> CY-3 floresan ışık üretir alkalin fosfataz için bir ticari kit ile doku bölümü geliştirin. </li> &lt;li&gt; 5 dakika boyunca yıkama tamponu ile bölüm yıkayın. Yıkama tamponu ile 5 dakika yıkama ve ardından, 5 dakika boyunca 2.5 ng / ml Hoechst boya, 100 ul Leke bölümü. </li><li> üreticinin protokolüne göre bir tampon montaj bölümüne monte edin. Doku Bölüm başına 15 ul kullanın ve doku ile aşırı montaj çözümü silin silin. sızıntı önlemek için şeffaf tırnak cilası ile slayt mühür. </li><li> 10X objektif yani toplam büyütme kullanarak bölüm floresan mikroskop gözlemleyin 100X olduğunu. </li></ol>

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There is nothing to disclose.

Bu çalışmanın amacı, in situ hibridizasyon miRNA kullanarak meme kanseri dokularında MiRNA ekspresyon seviyesini belirlemek için yapıldı. Deneyde, tüm koşullar meme kanseri dokusu için optimize olduğu not edilmelidir. Ayrıntılı optimizasyon diğer doku tipleri için gerekli olabilir. Bütün çözeltiler DEPC su ile yapılmalıdır ve kaplar DEPC işlenmiş, su ile durulandı ve daha sonra otoklavlanmalıdır kullanılır. Hatta hafif RNaz kirlenme deney nihai sonucu etkileyebilir. MiRNA çok k?...

Meme kanseri dünya genelinde kadın nüfusu etkileyen en yaygın maligniteler arasında yer alıyor. Meme kanseri 1.3 milyon vaka dünya çapında 1,2 yılda bildirilmektedir. Tümör hücreleri, geleneksel biyolojik homojen ve yüksek proliferatif olarak kabul edilmiş olmasına rağmen, meme kanseri genetik ve klinik heterojen olduğunu belirgin hale gelmiştir. Yaygın antikanser ilaçlara direnç kanser ilerleme ve mesafe metastazı 3 onun kolaylaştırılması yoluyla hastaların çoğunluğunda mortaliteye neden ilerlemiş meme kanserlerinin bir özelliğidir. MikroRNA&#39;lar (miRNA&#39;ların) mRNA translasyonunu bloke veya mRNA bozulmasını 4 aracılık gen ekspresyonunu düzenleyen yaklaşık 22 nükleotid uzunluğunda küçük RNA molekülleri sınıfıdır. 2.000 &#39;den fazla farklı miRNA&#39;lar kadar insan hastalıkları 5 ile bağlantılı olan, insanları tespit edilmiştir. çalışmalar artan bir dizi havE miRNA&#39;lar onkojenlerin ve tümörleri giderici olarak işlev görebilir ve genellikle tümör 6 düzensiz olduğunu göstermiştir. miRNA&#39;lar kuvvetle tümör hücre hareketi, invazyon ve metastaz 7 olanlarla birlikte hücre döngüsü ilerlemesinin, yaşlanma, apoptoz ve otofaji önemli regülatörleri kontrol ederek birincil tümörogenez tüm etkileyebilir. Aberrant miRNA ifade aynı zamanda sahne, farklılaşma, prognozu ve adjuvan tedavi 8 yanıt olarak klinik etkileri ile ilişkili olmuştur. Özellikle, miR-146 ifadesi akciğer kanserli hastalarda 9 kötü prognoz ile ilişkilidir arttı. Başka bir çalışmada, miRNA let7b kayıp sentezleme Sonuç olarak, zayıf yaşama 6, habis fenotipler ile bağlantılı ve göstermiştir. miRNA&#39;lar ifade eden hücrelerin ve yapı türlerini anlamak olduğunu mekaniğini anlamak önemli bir parçası geçtiği miRNA ifade etkisi hücrede değişiklikler veDoku 10 fenotip. stromal hücrelerde salgılandığı bulunmuştur belirli miRNA&#39;ların epitel hücreleri tarafından alınır ve özel olarak hedef doğrultusunda hareket edilir. Aynı şekilde, miR-212 ve miR-132 meme bezi gelişimi sırasında 11 duktal büyümesi için gerekli epitel-stromal etkileşimleri stroma tarafından salgılanan ve regüle edilir. Bu çalışmanın genel amacı in situ hibridizasyon miRNA ile meme kanseri dokularında miRNA ifade algılamak için ayrıntılı bir protokol açıklamaktır. Biz meme kanseri hasta örneklerinde miRNA 489 ifadesinin seviyelerini analiz etmek için tüm şartları optimize ettik. Bu amaçla, işaretlenmiş DIG eden deneyde nükleik asit (LNA) prob kilitli kullanılır. Bunu nükleotid bazı, yani, bir LNA modifikasyonu sahip 2 &#39;, oksijen atomu ve 4&#39; de hibridizasyon sonrası &quot;yerine kilitli&quot; kalacak şekilde nükleotidi giderir riboz omurga karbon atomu bağlayan bir metilen köprüsü. Bunun bir sonucu olarak, çok daha zorhibridize kompleksi 12,13 denatüre etmek için.

<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.

İnsan göğüs kanseri dokusu miRNA 489 ekspresyonunu belirlemek için kullanılmıştır. Meme bezi kanalı ve epitel hücreler bitişik iki hastada tümör dokusunda (Şekil 1A ve 1B) önemli ölçüde daha yüksek MiRNA-489 seviyelerini ifade bulunmuştur. Bu açıkça miRNA 489 ifadesi miRNA 489 tümör baskılayıcı aktivite düşündüren, tümör dokusunda kayıp olduğunu göstermektedir. daha da aynı hastadan alınan, bu sonuçlar, tümör do...

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.

miRNA kullanarak Meme Kanseri Dokularındaki miRNA İfade Klinikopatolojik Analizi<em&gt; In Situ</em&gt; Melezleme

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 Görüntüleme.  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.

Retrospektif mikroRNA sıralama: Parafin gömülü RNA Formalin sabit örnekler kullanarak Tamamlayıcı DNA Kütüphane hazırlık Protokolü

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

Genetik

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.

Doku özgü miRNA ifade profil oluşturma In Situ hibridizasyon fareyle kalp bölümleri içinde

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.

Metastatik kolorektal kanser hastalarında dolaşımdaki mikroRNA özel Panel tespiti

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

Kanser Araştırmaları

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.

Kantitatif Gerçek-zamanlı PCR (qPCR) tarafından Prostat tümörlerinde mikroRNA Algılama

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

Tıp

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.

Kombinatorik miRNA düzenleyen hücre döngüsü ve anjiogenezi tedavilere analizi

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.

Tümör hücrelerinde MicroRNA seviyeleri, fonksiyonları ve Ilişkili hedef genler değerlendirmek için bir In vitro protokol

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.

Meme Kanseri Hücrelerinde östrojen düzenlenmiş microRNA profil

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.

Meme kanseri arşivleme malzeme kullanarak bir Multiplex RNA temel ifade tahlil duruma getirilmesi

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

Biyomühendislik

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).

Prostat Kanseri Klinik Dokularındaki miRNA İfade Analizleri

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; Formalin Sabit Böbrek Dokular Hibridizasyon

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

miRNA kullanarak Meme Kanseri Dokularındaki miRNA İfade Klinikopatolojik Analizi

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