この作品は、カナダの癌協会研究所、助成金なしで資金を供給された。 019038。

<html> 1。前立腺のサンプルコレクション<ol><li>前立腺全摘除術時の前立腺のサンプルを収集します。標本は解剖学的ランドマークを用いて配向されている。前立腺と精嚢は、次のように描かれています。右側の緑、左側の青。 </li><li>前立腺のランダムな横方向の中央部には、直腸表面に垂直に撮影し、液体窒素中で凍結し、-80°C 9で保存されています。 </li><li>検体のバンクスライスは、複写指向（、前方後方、右と左）、quadrisectedです。セクションは、クライオスタットを用いてカットされます。 </li><li>セクションは、H＆Eで染色し、染色したスライドとそれに対応する画像上の腫瘍対正常な領域を決定し、記述するために病理学者によって検討されています。マークされた領域は、RNAは、以降の手順で抽出され、そこから腫瘍組織を抽出する領域を示すためのガイドとして使用されます。 </li></ol>&quot;ブレーク&gt; 2。サンプルからmiRNAを含むトータルRNAを単離<ol><li>ドライアイスと線引きコピーを参照の上に置いて凍結前立腺サンプルは、前立腺腫瘍のごく一部（50〜100mgの間）に切り取る。 </li><li> TRIzol試薬1mLに前立腺腫瘍組織をホモジナイズする。次の手順で数量がTRIzol試薬を1mLの使用に基づいています。 </li></ol>注：ここでは、RNAを抽出するためのTRIzol試薬を使用している、小さなRNAを含むトータルRNAを分離するが、他のキットを使用することもできます。 <ol start="3"><li>室温で5分間均質化し、サンプルをインキュベートします。 </li><li>サンプルにクロロホルム0.2 mLを加え、15秒間激しく振る。室温で3分間サンプルをインキュベートし、4℃で15分間12,000 xgで遠心分離℃、 </li><li>新鮮なチューブに無色の上部の水相を転送し、イソプロピルアルコール0.5mLを追加します。室温で10分間サンプルをインキュベートし、4℃で10分間12,000 xgで遠心分離℃、 </li><li> RNAを含有するペレットを乱すことなく、上清を注意深く吸引除去する。 75％エタノール1mLをRNAペレットを洗浄します。渦4で7500 xgで5分間遠心分離による試料と再堆積℃に</li><li>慎重に上清を吸引し、RNAペレットが完全に乾いていないことを確認し、5〜10分間RNAペレットを乾燥させます。ペレットのサイズに適切なヌクレアーゼフリー水に再溶解する。光度計1000分光光度計（260 nmと280 nmにおける吸光度を測定して）を使用してRNAの濃度を測定します。 </li><li>アジレントバイオアナライザを用いたRNAサンプルの品質と整合性を確認します。 </li></ol> 3。 RNAの逆転写<ol><li> RNAの逆転写は、メーカーの指示（キアゲン社）によるとmiScript逆転写キットを用いて行った。このキットには含まれています転写酵素およびポリ（A）ポリメラーゼを逆にしてください。 miScript RTバッファーは、Mg 2 +、dNTPを、oligo-dTプライマー、ランダムプライマーが含まれています。 </li><li> cDNAを合成するために10 pgのRNAの1μgの間に使用します。 RNAの1以上のμgを使用している場合は、適切なボリュームに直線的に反応をスケールアップ。 </li><li> 20μlの最終容量に反応をもたらすために5X miScript RTバッファー（4μl）を、miScript逆転写ミックス（1μL）、およびRNaseフリー水を含むマスターミックスを調製する。また、マスターミックスのテンプレートRNA（最大1μg）を含む。 </li><li> 37℃で60分間サンプルをインキュベート°C、95℃で5分間インキュベートした直後にこのステップは、ブロックまたは水浴を加熱し、PCRマシンで実行することができます。サーモは、最も適切かつ正確な方法があります。短期的に氷の上にcDNAを保存し、-20℃、長期保存用。 </li></ol> 4。検量線を作成する<ol><li> exper前にターゲットのmiRNAとiment、標準曲線は、そのクロッシングポイント（CP）（ 図2）に対して、既知濃度のcDNAを使用して生成されます。 </li><li> 2倍、10倍、50倍、250倍、1250倍の希釈液、目的の遺伝子の実質的な発現を有することが知られているサンプルの元のcDNAのシリーズを準備します。 </li><li>第5章 &quot;miRNAを検出するためのリアルタイムPCR&quot;で指定されたcDNAは、段階希釈しない静的な40倍希釈であることを修正して、PCRを実行します。 </li><li>あなたの標準曲線を生成するためにRelQuantソフトウェア（Roche）を用いて分析を実行します。 </li></ol>注：新しい標準曲線は、目的の各遺伝子を生成する必要があります。 5。 miRNAの検出のためのリアルタイムPCR <ol><li> miRNAのリアルタイムPCRは、メーカーの指示（キアゲン社）によるとmiScript SYBRグリーンPCR KitおよびmiScriptのPrimer Assayを用いて行った。を含むマスターミックスを調製2倍のQuantiTect SYBRグリーンPCRマスターミックス、10倍miScriptユニバーサルプライマー、10倍miScriptプライマーアッセイ、およびRNaseフリー水。 20μlの体積反応用のマスターミックスを調製する。 </li><li>プライマーアッセイは、興味のmiRNAに特異的である。再構成する10倍miScriptプライマーアッセイに、簡単にバイアルを遠心し、550μlのTEバッファー（pH 8.0）を追加します。 -20℃でボルテックス混合するために一時的にバイアル、小さいボリュームにアリコートプライマー、およびストア2つのプライマーを必要とされています。標的遺伝子とリファレンス遺伝子用のプライマーRNU6Bは、リファレンス遺伝子usedasです。 </li><li> -20℃でのcDNA 40倍と店舗余分なアリコートを希釈する</li><li> cDNAは、PCRのテンプレートとして機能します。 40倍希釈したcDNA2μlのを使用し、20μlのライトサイクラーキャピラリー（ロシュ）に分配する。 </li><li>各キャピラリーにマスターミックス18μlを追加し、キャピラリアダプタを使用して遠心します。 </li><li>そのようなサイクラー3のようなキャピラリーベースのリアルタイム·サイクラーに毛細血管を配置します。32キャピラリーカルーセル形式で5リアルタイムPCRシステム。 </li><li>次のようにPCRサイクリング·プログラムを実行します。 2倍のQuantiTect SYBRグリーンPCRマスターミックスでHotStarTaq Polymeraseを有効にするには、95℃でプレインキュベート15分間°C。 の50サイクルが続いています。 変性、15秒、94°C; アニーリング、30秒、55°C; 拡張子、30秒、70℃ </li><li>キャリブレーターになるサンプルを選択し、1に正規化され、その目標額を設定します。キャリブレータを他のすべてのサンプル中のmiRNAの相対的発現を比較します。 </li></ol>注：研究の中では、同じキャリブレーションサンプルは、結果の一貫性を維持するために使用されるべきである。 6。データの分析<ol><li> PCR反応の増幅曲線は、分子生化学のライトサイクラーソフトウェアバージョン3.5（ロシュ社）はグラフと数値で示されている。 、 &quot;定量化&quot;タブで反応を定量化するndはテキストフ​​ァイルにデータをエクスポートします。 </li><li>定量結果を生成するためにRelQuant解析ソフトウェア（ロシュ）にデータをインポートします。標的遺伝子、リファレンス遺伝子、および検量線のデータに別々のファイルをインポートします。 </li><li>ターゲットとリファレンス遺伝子の両方のキャリブレータの位置を指定します。また、サンプルの位置を指定します。データは、キャリブレータの比率を参照するには、ターゲットで割った値の異なるサンプルの比率を参照するターゲットとして表されます。以前に特定のmiRNAとハウスキーピング遺伝子用に生成された標準曲線は、未知の濃度のmiRNAのターゲットの定量的なデータを外挿するための参照標準として使用されます。 </li><li>三は、サンプルの複製され、グループとして分析し、濃度と三重の標準偏差が計算されることを意味しています。三連の1セットの残りの部分と矛盾している場合は、プログラムによって除外されます。 </li></ol> 7。代表的な結果 &lt;pのクラス= &quot;jove_content&quot;&gt;前立腺サンプルのqPCR解析の例を図3に示されています。結果はグラフィカルにだけでなく、数値的に描かれています。標的遺伝子は、miR-98の発現は約25℃のサイクル遅延増幅を示したリファレンス遺伝子の発現レベルを示すグラフ、U6は、周期約20℃で指数関数的増幅を開始します。この実験から得られたデータはテキストフ​​ァイルとしてエクスポートし、RelQuant解析ソフトウェアで解析した。キャリブレータとサンプルを含む毛細血管の位置が指定されています。 図4は、キャリブレータを1に設定し、校正器に対する相対他のサンプルの発現方法を示します。 <img src="/files/ftp_upload/3874/3874fig1.jpg" alt="図1"/> 図1。miScript逆転写反応とリアルタイムPCRの様々な手順を実行します。 <img src="/files/ftp_upload/3874/3874fig2.jpg" alt="図2"/> 図2標準曲線は、2倍、10倍、50倍、250倍、1250倍オリジナルのcDNAサンプルの希釈系列を使用して生成されます。 <img src="/files/ftp_upload/3874/3874fig3.jpg" alt="図3"/> 図3。ロシュ·ダイアグノスティックスのライトサイクラーソフトウェアは、グラフィカルとテキストによる実験の全体の情報が表示されます。定量的リアルタイムPCR増幅プロットは、異なるサンプルからの蛍光の増加を示しています。 <img src="/files/ftp_upload/3874/3874fig4.jpg" alt="図4"/> 図4のデータは、RelQuantライトサイクラー解析ソフトウェアを用いて定量した。通常は、3つのグループとして分析され、明確に矛盾した結果を生成するサンプルを除外し、三重の濃度と標準偏差が計算されることを意味されるサンプルのレプリケートされます。

<ol>
	<li>Reinhart, B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+in+plants.">MicroRNAs in plants.</a> Genes Dev. 16, 1616-1616 (2002).</li><li>Xu, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Drosophila+microRNA+Mir-14+suppresses+cell+death+and+is+required+for+normal+fat+metabolism.">The Drosophila microRNA Mir-14 suppresses cell death and is required for normal fat metabolism.</a> Curr. Biol. 13, 790 (2003).</li><li>Bueno, M. J., Perez, d. e. C. a. s. t. r. o., I, ., Malumbres, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+cell+proliferation+pathways+by+microRNAs.">Control of cell proliferation pathways by microRNAs.</a> Cell Cycle. 7, 3143-3143 (2008).</li><li>Friedman, R. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Most+mammalian+mRNAs+are+conserved+targets+of+microRNAs.">Most mammalian mRNAs are conserved targets of microRNAs.</a> Genome Res. 19, 92 (2009).</li><li>Bartel, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs:+genomics,+biogenesis,+mechanism,+and+function.">MicroRNAs: genomics, biogenesis, mechanism, and function.</a> Cell. 116, 281-281 (2004).</li><li>Croce, C. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Causes+and+consequences+of+microRNA+dysregulation+in+cancer.">Causes and consequences of microRNA dysregulation in cancer.</a> Nat. Rev. Genet. 10, 704 (2009).</li><li>Coppola, V., De, M. R., Bonci, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNAs+and+prostate+cancer.">MicroRNAs and prostate cancer.</a> Endocr. Relat. Cancer. 17, 1-1 (2010).</li><li>Gordanpour, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=miR-221+Is+down-regulated+in+TMPRSS2:ERG+fusion-positive+prostate+cancer.">miR-221 Is down-regulated in TMPRSS2:ERG fusion-positive prostate cancer.</a> Anticancer Res. 31, 403-403 (2011).</li><li>Nam, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expression+of+the+TMPRSS2:ERG+fusion+gene+predicts+cancer+recurrence+after+surgery+for+localised+prostate+cancer.">Expression of the TMPRSS2:ERG fusion gene predicts cancer recurrence after surgery for localised prostate cancer.</a> Br. J. Cancer. 97, 1690 (2007).</li><li>Ambs, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Genomic+profiling+of+microRNA+and+messenger+RNA+reveals+deregulated+microRNA+expression+in+prostate+cancer.">Genomic profiling of microRNA and messenger RNA reveals deregulated microRNA expression in prostate cancer.</a> Cancer Res. 68, 6162 (2008).</li><li>Liu, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+microRNA+miR-34a+inhibits+prostate+cancer+stem+cells+and+metastasis+by+directly+repressing+CD44.">The microRNA miR-34a inhibits prostate cancer stem cells and metastasis by directly repressing CD44.</a> Nat. Med. 17, 211 (2011).</li></ol>

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正常組織10に比較した場合、いくつかのmiRNAの異常な表現が一貫して前立腺腫瘍で発見されており、これらのmiRNAのいくつかは、前立腺癌11に対する潜在的な新規治療薬として名指しされている。したがって、miRNAの異常な発現レベルは、有用な診断および/または予後バイオマーカーにすることができます。ここで紹介するリアルタイム定量PCR手法は、前立腺腫瘍組織におけるm...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td></tr><tr><td> TRIzol試薬</td><td>インビトロジェン</td><td> 15596 </td></tr><tr><td> miScript逆転写キット</td><td>キアゲン</td><td> 218061 </td></tr><tr><td> miScriptプライマーアッセイ</td><td>キアゲン</td><td>特定の実験</td></tr><tr><td> miScript SYBRグリーンPCRキット</td><td>キアゲン</td><td> 218073 </td></tr><tr><td>ライトサイクラー3.5リアルタイムPCRシステム</td><td>ロシュ社</td><td></td></tr><tr><td>ライトサイクラーキャピラリー</td><td>ロシュ社</td><td> 04929292001 </td></tr><tr><td>光度計1000分光光度計</td><td>サーモフィッシャーサイエンティフィック</td><td> 2538 </td></tr><tr><td> Agilent 2100バイオアナライザ</td><td> G2943CA </td><td></td></tr></tbody></table>

microrna detection in prostate tumors by quantitative real-time pcr (qpcr)

マイクロRNA（miRNA）は、一本鎖、18から24ヌクレオチド長、非コーディングRNA分子である。彼らは、開発1、アポトーシス2、および細胞周期の調節3を含むほぼすべての細胞プロセスに関与している。 miRNAはそのターゲットメッセンジャーRNA（mRNA）5に結合することにより、ヒト遺伝子の30％〜90％4の発現を調節すると推定されている。 miRNAの広範囲の調節不全は、様々な疾患や癌のサブタイプ6で報告されています。その有病率とユニークな構造により、これらの小分子は、バイオマーカー、治療薬および/またはターゲットの次の世代になる可能性があります。 miRNAの発現を調べるために使われるメソッドは、SYBR Green I色素をベースと同様のTaqManプローブベース定量PCRが含まれています。 miRNAが効率的に臨床の現場で使用する場合は、それが新鮮な、そして/または、アーカイブされた臨床検体での検出は、正確な再現性、およびspであることが不可欠であるecific。定量PCRは、広くこのようなマイクロアレイ研究7などの全ゲノム解析におけるmiRNAの発現を検証するために使用されています。このプロトコルで使用されているサンプルは、臨床的限局性前立腺癌に対する根治的前立腺摘除術を施行した患者からのものであった。しかし、他の組織や細胞株は、切除した後に液体窒素で瞬間凍結した前立腺標本インチ置換することができます。臨床的変数とそれぞれの患者のフォローアップ情報は、その後の分析8に収集した。 前立腺腫瘍サンプル中のmiRNAレベルの定量化 。腫瘍の定量PCR解析の主な手順は次のとおりです。トータルRNA抽出、cDNA合成、およびmiRNA特異的プライマーを用いた定量PCR産物の検出。 mRNAは、miRNAの、および他のsmall RNAを含むトータルRNAは、TRIzol試薬を用いて検体から抽出した。キアゲンのmiScriptシステムはcDNAを合成し、定量PCR（ 図1）を実行するために使用されていました。内因性miRNAはPOではありません逆転写の過程で、したがって、lyadenylated、ポリ（A）ポリメラーゼがmiRNAをpolyadenylates。 miRNAはオリゴdTと逆転写酵素を用いてcDNAを合成するためのテンプレートとして使用されています。 oligo-dTプライマーの5 &#39;末端に普遍的なタグ配列は、PCRのステップでcDNAの増幅を容易にします。 PCR産物の増幅は、SYBRグリーン、二本鎖DNAにインターカレート色素が発する蛍光のレベルで検出されます。ユニバーサルタグ配列に結合するユニバーサルプライマーと一緒に特定のmiRNAのプライマーは、特定のmiRNAの配列を増幅します。 miScriptプライマーアッセイは、千以上の人間に固有のmiRNA、およびマウス固有のmiRNAの何百もご利用いただけます。相対定量法はmiRNAの発現を定量化するためにここで使用されていました。異なるサンプル間のばらつきを補正するために、ターゲットのmiRNAの発現レベルは、基準遺伝子の発現レベルに正規化されています。 GEの選択ターゲットの表現を正規化するのNEは、分析の相対定量法では非常に重要です。彼らは安定してほとんどのサンプルを越えて発現していることが考えられているとして、通常、この容量で使用されているリファレンス遺伝子の例としては、RNU6B、RNU44、とRNU48の小さなRNAである。このプロトコルでは、RNU6Bを参照遺伝子として使用されます。

Watch this Scientific Journal Video about MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR at JoVE.com

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

定量的リアルタイムポリメラーゼ連鎖反応（定量PCR）は、腫瘍サンプル中の様々なマイクロRNA（miRNA）分子の発現レベルを調査するために迅速かつ高感度な方法です。異なるmiRNA分子の何百ものこのメソッド式を使用して、増幅定量化し、同じcDNAをテンプレートから分析することができます。

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 ...

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

24.4K ビュー.  University of Toronto. 定量的リアルタイムポリメラーゼ連鎖反応（定量PCR）は、腫瘍サンプル中の様々なマイクロRNA（miRNA）分子の発現レベルを調査するために迅速かつ高感度な方法です。異なるmiRNA分子の何百ものこのメソッド式を使用して、増幅定量化し、同じcDNAをテンプレートから分析することができます。

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

Multispectral Real-time Fluorescence Imaging for Intraoperative Detection of the Sentinel Lymph Node in Gynecologic Oncology

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical excision of the tumor with a full lymphadenectomy in the drainage area of the tumor. However, removal of lymph nodes is associated with increased morbidity due to infection, wound breakdown and lymphedema.4,5 As an alternative, the sentinel lymph node procedure (SLN) was developed several decades ago to detect the first draining lymph node from the tumor.6 In case of lymphogenic dissemination, the SLN is the first lymph node that is affected (Figure 1). Hence, if the SLN does not contain metastases, downstream lymph nodes will also be free from tumor metastases and need not to be removed. The SLN procedure is part of the treatment for many tumor types, like breast cancer and melanoma, but also for cancer of the vulva and cervix.7 The current standard methodology for SLN-detection is by peritumoral injection of radiocolloid one day prior to surgery, and a colored dye intraoperatively. Disadvantages of the procedure in cervical and vulvar cancer are multiple injections in the genital area, leading to increased psychological distress for the patient, and the use of radioactive colloid.
Multispectral fluorescence imaging is an emerging imaging modality that can be applied intraoperatively without the need for injection of radiocolloid. For intraoperative fluorescence imaging, two components are needed: a fluorescent agent and a quantitative optical system for intraoperative imaging. As a fluorophore we have used indocyanine green (ICG). ICG has been used for many decades to assess cardiac function, cerebral perfusion and liver perfusion.8 It is an inert drug with a safe pharmaco-biological profile. When excited at around 750 nm, it emits light in the near-infrared spectrum around 800 nm. A custom-made multispectral fluorescence imaging camera system was used.9.
The aim of this video article is to demonstrate the detection of the SLN using intraoperative fluorescence imaging in patients with cervical and vulvar cancer. Fluorescence imaging is used in conjunction with the standard procedure, consisting of radiocolloid and a blue dye. In the future, intraoperative fluorescence imaging might replace the current method and is also easily transferable to other indications like breast cancer and melanoma.

Fluorescence imaging is a promising innovative modality for image-guided surgery in surgical oncology. In this video we describe the technical procedure for detection of the sentinel lymph node using fluorescence imaging as showcased in gynecologic oncologicy. A multispectral fluorescence camera system, together with the fluorescent agent indocyanine green, is applied.

婦人科腫瘍におけるセンチネルリンパ節の術中検出のためのマルチスペクトルのリアルタイム蛍光イメージング

The prognosis in virtually all solid tumors depends on the presence or absence of lymph node metastases.1-3 Surgical treatment most often combines radical ...

医学

A Real-time Electrical Impedance Based Technique to Measure Invasion of Endothelial Cell Monolayer by Cancer Cells

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of endothelial junctions and finally invasion and migration of tumor cells through the endothelial layer to enter the bloodstream as a means of transport to distant sites in the host1-3. Once in the circulatory system, cancer cells adhere to capillary walls and extravasate to the surrounding tissue to form metastatic tumors4,5. The various components of tumor cell-endothelial cell interaction can be replicated in vitro by challenging a monolayer of human umbilical vein endothelial cells (HUVEC) with cancer cells. Studies performed with electron and phase-contrast microscopy suggest that the in vitro sequence of events fairly represent the in vivo metastatic process6. Here, we describe an electrical-impedance based technique that monitors and quantifies in real-time the invasion of endothelial cells by malignant tumor cells.
Giaever and Keese first described a technique for measuring fluctuations in impedance when a population of cells grow on the surface of electrodes7,8. The xCELLigence instrument, manufactured by Roche, utilizes a similar technique to measure changes in electrical impedance as cells attach and spread in a culture dish covered with a gold microelectrode array that covers approximately 80% of the area on the bottom of a well. As cells attach and spread on the electrode surface, it leads to an increase in electrical impedance9-12. The impedance is displayed as a dimensionless parameter termed cell-index, which is directly proportional to the total area of tissue-culture well that is covered by cells. Hence, the cell-index can be used to monitor cell adhesion, spreading, morphology and cell density.
The invasion assay described in this article is based on changes in electrical impedance at the electrode/cell interphase, as a population of malignant cells invade through a HUVEC monolayer (Figure 1). The disruption of endothelial junctions, retraction of endothelial monolayer and replacement by tumor cells lead to large changes in impedance. These changes directly correlate with the invasive capacity of tumor cells, i.e., invasion by highly aggressive cells lead to large changes in cell impedance and vice versa. This technique provides a two-fold advantage over existing methods of measuring invasion, such as boyden chamber and matrigel assays: 1) the endothelial cell-tumor cell interaction more closely mimics the in vivo process, and 2) the data is obtained in real-time and is more easily quantifiable, as opposed to end-point analysis for other methods.

This article describes an in vitro technique for monitoring cancer cells invading through a monolayer of endothelial cells. The data is acquired in real-time as a function of changes in impedance on the surface of electrodes at the well bottom.

癌細胞による内皮細胞単層の侵入を測定するリアルタイム電気インピーダンス基づく手法

Metastatic dissemination of malignant cells requires degradation of basement membrane, attachment of tumor cells to vascular endothelium, retraction of ...

Evaluation of Nanoparticle Uptake in Tumors in Real Time Using Intravital Imaging

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle uptake in tumors at the microscopic level1,2,3, highlighting the utility of a suitable xenograft model in which to perform detailed uptake analyses. Here, we use high-resolution intravital imaging to evaluate nanoparticle uptake in human tumor xenografts in a modified, shell-less chicken embryo model. The chicken embryo model is particularly well-suited for these in vivo analyses because it supports the growth of human tumors, is relatively inexpensive and does not require anesthetization or surgery 4,5. Tumor cells form fully vascularized xenografts within 7 days when implanted into the chorioallantoic membrane (CAM) 6. The resulting tumors are visualized by non-invasive real-time, high-resolution imaging that can be maintained for up to 72 hours with little impact on either the host or tumor systems. Nanoparticles with a wide range of sizes and formulations administered distal to the tumor can be visualized and quantified as they flow through the bloodstream, extravasate from leaky tumor vasculature, and accumulate at the tumor site. We describe here the analysis of nanoparticles derived from Cowpea mosaic virus (CPMV) decorated with near-infrared fluorescent dyes and/or polyethylene glycol polymers (PEG) 7, 8, 9,10,11. Upon intravenous administration, these viral nanoparticles are rapidly internalized by endothelial cells, resulting in global labeling of the vasculature both outside and within the tumor7,12. PEGylation of the viral nanoparticles increases their plasma half-life, extends their time in the circulation, and ultimately enhances their accumulation in tumors via the enhanced permeability and retention (EPR) effect 7, 10,11. The rate and extent of accumulation of nanoparticles in a tumor is measured over time using image analysis software. This technique provides a method to both visualize and quantify nanoparticle dynamics in human tumors.

We present a novel approach to quantify nanoparticle localization in the vasculature of human xenografted tumors using dynamic, real-time intravital imaging in an avian embryo model.

生体内イメージングを使ってリアルタイムで腫瘍におけるナノ粒子の取り込みの評価

Current technologies for tumor imaging, such as ultrasound, MRI, PET and CT, are unable to yield high-resolution images for the assessment of nanoparticle ...

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of hospitalizations in children with a public health impact similar to that of Rotavirus. NoVs are RNA viruses of great genetic diversity and there is a continuous appearance of new strains. Five genogroups are recognized; GI and GII with their many genotypes and subtypes being the most important for human infection. However, the diagnosis of these two genotypes remains problematic, delaying diagnosis and treatment. 1, 2, 3
For RNA extraction from stool specimens the most commonly used method is the QIAmp Viral RNA commercial kit from Qiagen. This method combines the binding properties of a silica gel membrane, buffers that control RNases and provide optimum binding of the RNA to the column together with the speed of microspin. This method is simple, fast and reliable and is carried out in a few steps that are detailed in the description provided by the manufacturer. 
 Norovirus is second only to rotavirus as the most common cause of diarrhea. Norovirus diagnosis should be available in all studies on pathogenesis of diarrhea as well as in outbreaks or individual diarrhea cases. At present however norovirus diagnosis is restricted to only a few centers due to the lack of simple methods of diagnosis. This delays diagnosis and treatment 1, 2, 3. In addition, due to costs and regulated transportation of corrosive buffers within and between countries use of these manufactured kits poses logistical problems. As a result, in this protocol we describe an alternative, economic, in-house method which is based on the original Boom et al. method4 which uses the nucleic acid binding properties of silica particles together with the anti-nuclease properties of guanidinium thiocyanate.
For the detection and genogrouping (GI and GII) of NoVs isolates from stool specimens, several RT-PCR protocols utilizing different targets have been developed. The consensus is that an RT-PCR using TaqMan chemistry would be the best molecular technique for diagnosis, because it combines high sensitivity, specificity and reproducibility with high throughput and ease of use. Here we describe an assay targeting the open reading frame 1 (ORF1)-ORF2 junction region; the most conserved region of the NoV genome and hence most suitable for diagnosis. For further genetic analysis a conventional RT-PCR that targets the highly variable N-terminal-shell from the major protein of the capsid (Region C) using primers originally described by Kojima et al. 5 is detailed. Sequencing of the PCR product from the conventional PCR enables the differentiation of genotypes belonging to the GI and GII genogroups.

Detection and Genogrouping of Noroviruses from Children&#039;s Stools By Taqman One-step RT-PCR

A One-Step RT-PCR assay for detection and genogroup identification of Norovirus isolates from children&#x2019;s stools, that utilizes primers and TaqMan probes specific to the open reading frame 1 (ORF1)-ORF2 junction region, the most conserved region of the Norovirus genome is described. A non-commercial, cost-effective RNA extraction method is detailed.

のTaqManワンステップRT-PCR法によるキッズスツールからノロウイルスの検出とGenogrouping

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

Heterogeneity Mapping of Protein Expression in Tumors using Quantitative Immunofluorescence

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of areas of distinct differentiation into recognized histological subtypes, or different pathological grade, often there are more subtle differences in phenotype which defy accurate classification (Figure 1). Ultimately, since morphology is dictated by the underlying molecular phenotype, areas with visible differences are likely to be accompanied by differences in the expression of proteins which orchestrate cellular function and behavior, and therefore, appearance. The significance of visible and invisible (molecular) heterogeneity for prognosis is unknown, but recent evidence suggests that, at least at the genetic level, heterogeneity exists in the primary tumor1,2, and some of these sub-clones give rise to metastatic (and therefore lethal) disease. 
Moreover, some proteins are measured as biomarkers because they are the targets of therapy (for instance ER and HER2 for tamoxifen and trastuzumab (Herceptin), respectively). If these proteins show variable expression within a tumor then therapeutic responses may also be variable. The widely used histopathologic scoring schemes for immunohistochemistry either ignore, or numerically homogenize the quantification of protein expression. Similarly, in destructive techniques, where the tumor samples are homogenized (such as gene expression profiling), quantitative information can be elucidated, but spatial information is lost. Genetic heterogeneity mapping approaches in pancreatic cancer have relied either on generation of a single cell suspension3, or on macrodissection4. A recent study has used quantum dots in order to map morphologic and molecular heterogeneity in prostate cancer tissue5, providing proof of principle that morphology and molecular mapping is feasible, but falling short of quantifying the heterogeneity. Since immunohistochemistry is, at best, only semi-quantitative and subject to intra- and inter-observer bias, more sensitive and quantitative methodologies are required in order to accurately map and quantify tissue heterogeneity in situ.
We have developed and applied an experimental and statistical methodology in order to systematically quantify the heterogeneity of protein expression in whole tissue sections of tumors, based on the Automated QUantitative Analysis (AQUA) system6. Tissue sections are labeled with specific antibodies directed against cytokeratins and targets of interest, coupled to fluorophore-labeled secondary antibodies. Slides are imaged using a whole-slide fluorescence scanner. Images are subdivided into hundreds to thousands of tiles, and each tile is then assigned an AQUA score which is a measure of protein concentration within the epithelial (tumor) component of the tissue. Heatmaps are generated to represent tissue expression of the proteins and a heterogeneity score assigned, using a statistical measure of heterogeneity originally used in ecology, based on the Simpson's biodiversity index7.
To date there have been no attempts to systematically map and quantify this variability in tandem with protein expression, in histological preparations. Here, we illustrate the first use of the method applied to ER and HER2 biomarker expression in ovarian cancer. Using this method paves the way for analyzing heterogeneity as an independent variable in studies of biomarker expression in translational studies, in order to establish the significance of heterogeneity in prognosis and prediction of responses to therapy.

Here we describe a method to quantify molecular heterogeneity in histological sections of tumor material using quantitative immunofluorescence, image analysis, and a statistical measure of heterogeneity. The method is intended for use in clinical biomarker development and analysis.

定量的免疫蛍光法を用いて腫瘍におけるタンパク質発現の不均一性のマッピング

Morphologic heterogeneity within an individual tumor is well-recognized by histopathologists in surgical practice. While this often takes the form of ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

の同定<em&gt;眠れる森の美女</emリンカ媒介PCRを用いた固形腫瘍&gt;トランスポゾン挿入

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Movement Retraining using Real-time Feedback of Performance

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular patterns responsible for governing the movement performance. This motor learning can be enhanced through a number of methods that are utilized in research and clinical settings alike. In general, verbal feedback of performance in real-time or knowledge of results following movement is commonly used clinically as a preliminary means of instilling motor learning. Depending on patient preference and learning style, visual feedback (e.g. through use of a mirror or different types of video) or proprioceptive guidance utilizing therapist touch, are used to supplement verbal instructions from the therapist. Indeed, a combination of these forms of feedback is commonplace in the clinical setting to facilitate motor learning and optimize outcomes.
Laboratory-based, quantitative motion analysis has been a mainstay in research settings to provide accurate and objective analysis of a variety of movements in healthy and injured populations. While the actual mechanisms of capturing the movements may differ, all current motion analysis systems rely on the ability to track the movement of body segments and joints and to use established equations of motion to quantify key movement patterns. Due to limitations in acquisition and processing speed, analysis and description of the movements has traditionally occurred offline after completion of a given testing session.
This paper will highlight a new supplement to standard motion analysis techniques that relies on the near instantaneous assessment and quantification of movement patterns and the display of specific movement characteristics to the patient during a movement analysis session. As a result, this novel technique can provide a new method of feedback delivery that has advantages over currently used feedback methods.

Retraining abnormal movement patterns following injury or disease is a key component of physical rehabilitation. Recent advances in technology have permitted accurate assessment of movement during a variety of tasks, with near instantaneous quantification of results. This provides new opportunities for modification of faulty movement patterns in real time.

パフォーマンスのリアルタイムのフィードバックを使用して再教育運動

Any modification of movement - especially movement patterns that have been honed over a number of years - requires re-organization of the neuromuscular ...

Using Quantitative Real-time PCR to Determine Donor Cell Engraftment in a Competitive Murine Bone Marrow Transplantation Model

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules that regulate HSCs. In these transplant model systems, the function of HSCs is determined by the ability of these cells to engraft and reconstitute lethally irradiated recipient mice. Commonly, the donor cell contribution/engraftment is measured by antibodies to donor- specific cell surface proteins using flow cytometry. However, this method heavily depends on the specificity and the ability of the cell surface marker to differentiate donor-derived cells from recipient-originated cells, which may not be available for all mouse strains. Considering the various backgrounds of genetically modified mouse strains in the market, this cell surface/ flow cytometry-based method has significant limitations especially in mouse strains that lack well-defined surface markers to separate donor cells from congenic recipient cells. Here, we reported a PCR-based technique to determine donor cell engraftment/contribution in transplant recipient mice. We transplanted male donor bone marrow HSCs to lethally irradiated congenic female mice. Peripheral blood samples were collected at different time points post transplantation. Bone marrow samples were obtained at the end of the experiments. Genomic DNA was isolated and the Y chromosome specific gene, Zfy1, was amplified using quantitative Real time PCR. The engraftment of male donor-derived cells in the female recipient mice was calculated against standard curve with known percentage of male vs. female DNAs. Bcl2 was used as a reference gene to normalize the total DNA amount. Our data suggested that this approach reliably determines donor cell engraftment and provides a useful, yet simple method in measuring hematopoietic cell reconstitution in murine bone marrow transplantation models. Our method can be routinely performed in most laboratories because no costly equipment such as flow cytometry is required.

Determining donor cell engraftment presents a challenge in mouse bone marrow transplant models that lack well-defined phenotypical markers. We described a methodology to quantify male donor cell engraftment in female transplant recipient mice. This method can be used in all mouse strains for the study of HSC functions.

競争力のあるマウス骨髄移植モデルにおけるドナー細胞の生着を​​決定するために、定量的リアルタイムPCRを用いて

Murine bone marrow transplantation models provide an important tool in measuring hematopoietic stem cell (HSC) functions and determining genes/molecules ...

Cerebrospinal Fluid MicroRNA Profiling Using Quantitative Real Time PCR

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their translational repression. Changes in miRNA expression have been shown to be involved in the development of all major complex diseases. Furthermore, recent findings showed that miRNAs can be secreted to the extracellular environment and enter the bloodstream and other body fluids where they can circulate with high stability. The function of such circulating miRNAs remains largely elusive, but systematic high throughput approaches, such as miRNA profiling arrays, have lead to the identification of miRNA signatures in several pathological conditions, including neurodegenerative disorders and several types of cancers. In this context, the identification of miRNA expression profile in the cerebrospinal fluid, as reported in our recent study, makes miRNAs attractive candidates for biomarker analysis.
There are several tools available for profiling microRNAs, such as microarrays, quantitative real-time PCR (qPCR), and deep sequencing. Here, we describe a sensitive method to profile microRNAs in cerebrospinal fluids by quantitative real-time PCR. We used the Exiqon microRNA ready-to-use PCR human panels I and II V2.R, which allows detection of 742 unique human microRNAs. We performed the arrays in triplicate runs and we processed and analyzed data using the GenEx Professional 5 software.
Using this protocol, we have successfully profiled microRNAs in various types of cell lines and primary cells, CSF, plasma, and formalin-fixed paraffin-embedded tissues.

We describe a protocol of real time PCR to profile microRNAs in the cerebrospinal fluid (CSF). With the exception of RNA extraction protocols, the procedure can be extended to RNA extracted from other body fluids, cultured cells, or tissue specimens.

定量リアルタイムPCRを用いて脳脊髄液マイクロRNAプロファイリング

MicroRNAs (miRNAs) constitute a potent layer of gene regulation by guiding RISC to target sites located on mRNAs and, consequently, by modulating their ...

Real-time Cytotoxicity Assays in Human Whole Blood

A live cell-based whole blood cytotoxicity assay (WCA) that allows access to temporal information of the overall cell cytotoxicity is developed with high-throughput cell positioning technology. The targeted tumor cell populations are first preprogrammed to immobilization into an array format, and labeled with green fluorescent cytosolic dyes. Following the cell array formation, antibody drugs are added in combination with human whole blood. Propidium iodide (PI) is then added to assess cell death. The cell array is analyzed with an automatic imaging system. While cytosolic dye labels the targeted tumor cell populations, PI labels the dead tumor cell populations. Thus, the percentage of target cancer cell killing can be quantified by calculating the number of surviving targeted cells to the number of dead targeted cells. With this method, researchers are able to access time-dependent and dose-dependent cell cytotoxicity information. Remarkably, no hazardous radiochemicals are used. The WCA presented here has been tested with lymphoma, leukemia, and solid tumor cell lines. Therefore, WCA allows researchers to assess drug efficacy in a highly relevant ex&#160;vivo condition.

The whole blood cytotoxicity assay (WCA) is a cytotoxicity assay developed by incorporating high-throughput cell positioning technology with fluorescence microscopy and automated image processing. Here, we describe how lymphoma cells treated with an anti-CD20 antibody can be analyzed real-time in human whole blood to provide quantitative cellular cytotoxicity analysis.

ヒト全血でのリアルタイム細胞毒性アッセイ

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