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.

この研究は、全南大学校和順病院、サウスカロライナ大学の治験審査委員会によって承認されました。乳がんと一致した隣接する正常な乳房組織で構成されるTMAサンプルは全南大学校和順病院、韓国バイオバンクネットワークのメンバーのバイオバンクから提供されました。 1.溶液の調製<ol><li> DNアーゼおよびRNaseフリー水1リットルを準備します。ジエチル（DEPC）処理水を使用して、すべての化学溶液を作ります。 DEPCの500μlの水1Lを治療し、室温で3時間インキュベートします。 DEPCを不活性化するために水をオートクレーブ。 注意：それは発癌物質知られているので、DEPCを吸入しないでください。 </li><li> DEPC処理水を使用して、10×PBSから1×PBSを準備します。 </li><li> 、20×SSC（塩化ナトリウムの175.3グラムと800ミリリットルの水でクエン酸ナトリウムの88.2グラムを溶解を準備HClを14 N溶液数滴でpHを7.0に調整し、ワットで1Lにボリュームを調整ER）、4％パラホルムアルデヒド、95％エタノール、80％エタノールDEPC処理水を用いました。 </li><li> 8トリmlのDEPC処理水590ミリリットル中のHClの1.05ミリリットルを加え、アセチル化のための無水酢酸1.5ミリリットルを追加します。 </li><li>バッファ中の50μg/μlのプロテ​​イナーゼK（50 mMトリス - 塩酸、pHが8.0をDEPCで処理水を）を準備します。 </li><li>ホルムアミド500μlの、20×SSCを250μl、50×デンハルト溶液100μl、T-RNAの12.5μlを、ニシン精子DNA2.5μlの、RVCの30μlの、ブロッキング試薬及び50μlに0.02グラムで構成されるハイブリダイゼーション緩衝液を準備しますDEPC水の。新鮮な準備をします。 </li></ol> 2.乳房組織切片<ol><li>ミクロトームで5μmのホルマリン固定パラフィン包埋切片をカットし、スライド14を充電するためのセクションを適用します。 注：ここで使用される乳房組織は、韓国バイオバンクネットワークから先に調製しました。 </li></ol> 3.組織標本<ol><li>あちこちパラフィンを削除します10分間新鮮なキシレンでコプリンジャーの2倍に各々のスライドを浸漬することにより、組織をMとのddH 2 Oにエタノール（100％、95％及び80％）の濃度を減少させるで5分間のインキュベーションによって組織切片を再水和ドラフト内で、この手順を実行します。 </li><li> 5分間のDEPC処理水で組織を浸します。この時点で、疎水性バリアペンで組織切片の周りに境界線を作ります。 </li><li> PBSで洗浄した15分間、4％PFAで組織を修正しました。 </li><li> PBSで洗浄し、続いて10分間、0.3％トリトンX-100 / PBSで組織を浸します。 </li><li> 37℃で15分間の50μg/μlのプロテ​​イナーゼK溶液で試料をインキュベートします。その後PBSで洗浄し、続いて室温で5分間、トリエタノールアミンおよび無水酢酸で組織切片をインキュベートします。いいえPBS洗浄は、プロテイナーゼK処理およびトリエタノールアミン/無水酢酸処理の間に必要とされません。 </li></ol> 4.ハイブリダイゼーション<ol><li>徹底的にincu組織を洗います室温で2〜3時間のためのハイブリダイゼーション緩衝液を用いBATE組織切片。典型的には、120μlのハイブリダイゼーション緩衝液は、組織切片をカバーするのに十分です。 </li><li>希釈5&#39;-DIGは、120μlのハイブリダイゼーション溶液中の午後3時の最終濃度にLNAプローブを標識しました。ストック濃度は、通常、40 ngの/μlであるように、120μlのハイブリダイゼーション緩衝液に株式の3μlを添加し、95℃で5分間混合物をインキュベートします。 </li><li>加湿チャンバー内で54°C（Tm）がでプローブ一晩で組織切片をインキュベートします。典型的には、120μlの1部をカバーするのに十分です。 2湿ったペーパータオルを使用して湿度を作成します。室の理想的なサイズは8インチ×10インチです。 </li><li>注意：Tmは異なるmiRNAのために異なる場合があります。 54°Cは、miR-489のための言及です。 </li></ol> 5.ジェンウォッシュ<ol><li> 7分間の5倍のSSCで組織を洗浄（ハイブリダイゼーション工程の後に、RNaseを含まない条件がもはや必須ではありません）。 </li><li>組織切片のウィットを洗います二回57℃で7分間の時間1×SSC。 </li><li> 57℃で7分間、0.2×のSSCで組織切片を洗浄します。 </li><li>室温で7分間、0.2×のSSCで組織切片を洗浄します。 </li><li> 10分間、PBS中の組織切片をインキュベートします。 </li></ol> 6.ブロッキング<ol><li>バッファは、PBSの18メートルで、BSAのTween-20を3μlの10μlのを2ミリリットルのFBSを追加ブロッキングようにするには（湿潤チャンバー内で1時間室温でブロッキング緩衝液で組織切片をインキュベートします。Storeは4でブロッキング緩衝液 °C）。 </li></ol> 7.一次抗体インキュベーション開発<ol><li>ブロッキング緩衝液中に300抗DIG-AP抗体と4℃で一晩抗体を用いた組織切片をインキュベート：1に希釈します。 </li><li> 5分ごと（開発キットにより提供バッファ）のために、洗浄緩衝液で3回のセクションを洗ってください。 </li><li> CY-3蛍光光を生成するアルカリホスファターゼのための市販のキットを用いて組織切片を開発します。 </li> &lt;李&gt;は5分間の洗浄バッファーでセクションを洗ってください。洗浄緩衝液で5分間洗浄した5分間2.5 ngの/μlのヘキスト染料を100μlとステインセクション。 </li><li>製造業者のプロトコルに従ってバッファを搭載してセクションをマウントします。組織切片あたり15μLを使用して、組織拭くと、過剰なマウントソリューションを拭いてください。漏れを防止するために、明確なマニキュアでスライドを密閉します。 </li><li>トータルの倍率は100倍である10倍レンズを使用したセクションの蛍光顕微鏡を観察します。 </li></ol>

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There is nothing to disclose.

この研究の目的は、in situハイブリダイゼーションのmiRNAを用いた乳癌組織におけるmiRNA発現のレベルを決定することでした。なお、この実験では、全ての条件が乳癌組織のために最適化されたことに注意しなければなりません。さらなる最適化は、他の組織タイプのために必要になることがあります。全ての溶液は、DEPC水で行う必要があり、使用されるすべての容器はDEPC処理水ですす...

乳がんは世界中の女性人口に影響を与える最も一般的な悪性腫瘍の一つです。乳癌の130万以上の例は世界的に毎年1,2報告されています。腫瘍細胞は、伝統的に、生物学的に均一で高い増殖性とみなされてきたが、乳癌は遺伝的および臨床的に不均一であることが明らかになってきています。流行している抗がん剤に対する耐性は、癌の進行と距離転移3のその促進を通じ、大多数の患者の死亡率につながる、先進的な乳癌の特徴です。 マイクロRNA（miRNA）はmRNAの翻訳を遮断またはmRNA分解4を媒介することにより遺伝子発現を調節する約22ヌクレオチド長の小RNA分子のクラスです。 2,000人以上の異なるmiRNAは、これまでヒト疾患5にリンクされている人間、で同定されています。研究の増加アレイは、肝炎Eは、miRNAが癌遺伝子及び腫瘍抑制因子として機能することができ、多くの場合、腫瘍6の異常調節されていることを実証しました。 miRNAは強く腫瘍細胞の運動性、浸潤および転移7のものと一緒に、細胞周期の進行、老化、アポトーシスおよびオートファジーの主要な調節因子を制御することにより、一次腫瘍形成のすべてに影響を与えます。 異常なmiRNA発現はまた、ステージ、分化、予後およびアジュバント療法8に対する応答として臨床的意義と関連しています。具体的には、のmiR-146の発現が、 ​​肺癌患者9における予後不良と相関して増加しました。別の研究では、miRNA-let7bの失われた式は、悪性の表現型にリンクされていると、結果的に、低い生存率6されていることを実証しました。 miRNAを発現する細胞と構造の種類を理解することは、miRNAの発現に影響を​​与える細胞の変化、それを通して力学を理解することの重要な部分であり、組織は10を表現型。間質細胞で分泌されることがわかっ特定のmiRNAは、上皮細胞によって取り込まれ、具体的にその標的に作用しています。同じ静脈では、のmiR-212およびmiR-132は、間質から分泌され、乳腺発達11時の乳管伸長に必要な上皮-間質相互作用を調節しています。この論文の全体的な目的は、in situハイブリダイゼーションにおける miRNAを介して乳癌組織におけるmiRNAの発現を検出するための詳細なプロトコルを説明することです。我々は、乳癌患者サンプル中のmiRNA-489の発現のレベルをアッセイするために、すべての条件を最適化しました。この目的のために、我々は、DIGがロックされた核酸（LNA）、我々の実験でプローブを標識しました。そのヌクレオチドのいくつかは、それは、2 &#39;酸素原子と4&#39;は、ハイブリダイゼーション後の「適所にロックされた」ままになるようにヌクレオチドを​​固定リボース骨格の炭素原子を連結するメチレン架橋であり、LNA修飾を有していました。結果として、それははるかに困難ですハイブリダイズした複合体は、12,13を変性するために。

<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.

ヒト乳癌組織のmiRNA-489の発現を決定しました。乳腺管上皮細胞は、隣接する二人の患者における腫瘍組織（ 図1Aおよび1B）よりも有意に高かったmiRNA-489レベルを発現することが見出されました。これは明らかなmiRNA-489の発現がmiRNA-489の腫瘍抑制活性を示唆し、腫瘍組織では失われていることを示しています。さらに、これらの結果を確認する?...

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を用いた乳がん組織におけるmiRNA発現の臨床病理学的解析<em&gt;その場で</em&gt;ハイブリダイゼーション

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

ビデオ: 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.

ホルマリン固定パラフィン包埋 RNA 試料を用いた回顧マイクロ Rna シーケンス: 相補的な DNA ライブラリの準備のプロトコル

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

遺伝学

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.

マウス心臓のセクションを使用してその場で交配で発現組織固有の miRNA

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.

転移性大腸癌患者における循環マイクロ Rna カスタム パネルの検出

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

がん研究

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.

定量的リアルタイムPCR（定量PCR）による前立腺腫瘍におけるマイクロRNAの検出

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

医学

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.

組合せ miRNA 細胞周期の調節、血管新生治療の解析

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.

腫瘍細胞におけるマイクロRNAレベル、機能、および関連する標的遺伝子を評価するためのインビトロプロトコル

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.

乳癌細胞におけるエストロゲン調節マイクロRNAのプロファイリング

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.

乳房癌のアーカイブ材料を用いた多重 RNA に基づく発現アッセイの最適化

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

生物工学

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).

前立腺癌臨床組織におけるmiRNA発現解析

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.

マイクロRNA<em&gt;その場で</em&gt;ホルマリン固定腎臓組織のためのハイブリダイゼーション

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

miRNAを用いた乳がん組織におけるmiRNA発現の臨床病理学的解析

要約

さらに動画を探す

この動画の章

ホルマリン固定パラフィン包埋 RNA 試料を用いた回顧マイクロ Rna シーケンス: 相補的な DNA ライブラリの準備のプロトコル

マウス心臓のセクションを使用してその場で交配で発現組織固有の miRNA

転移性大腸癌患者における循環マイクロ Rna カスタムパネルの検出

定量的リアルタイムPCR（定量PCR）による前立腺腫瘍におけるマイクロRNAの検出

組合せ miRNA 細胞周期の調節、血管新生治療の解析

腫瘍細胞におけるマイクロRNAレベル、機能、および関連する標的遺伝子を評価するためのインビトロプロトコル

乳癌細胞におけるエストロゲン調節マイクロRNAのプロファイリング

乳房癌のアーカイブ材料を用いた多重 RNA に基づく発現アッセイの最適化

前立腺癌臨床組織におけるmiRNA発現解析

マイクロRNA

miRNAを用いた乳がん組織におけるmiRNA発現の臨床病理学的解析

要約

さらに動画を探す

この動画の章

ホルマリン固定パラフィン包埋 RNA 試料を用いた回顧マイクロ Rna シーケンス: 相補的な DNA ライブラリの準備のプロトコル

マウス心臓のセクションを使用してその場で交配で発現組織固有の miRNA

転移性大腸癌患者における循環マイクロ Rna カスタム パネルの検出

定量的リアルタイムPCR（定量PCR）による前立腺腫瘍におけるマイクロRNAの検出

組合せ miRNA 細胞周期の調節、血管新生治療の解析

腫瘍細胞におけるマイクロRNAレベル、機能、および関連する標的遺伝子を評価するためのインビトロプロトコル

乳癌細胞におけるエストロゲン調節マイクロRNAのプロファイリング

乳房癌のアーカイブ材料を用いた多重 RNA に基づく発現アッセイの最適化

前立腺癌臨床組織におけるmiRNA発現解析

マイクロRNA

転移性大腸癌患者における循環マイクロ Rna カスタムパネルの検出