著者らは、NOVのための標準と制御陽性の親切な贈り物を疾病管理予防のための国立カリシウイルス研究所センター（CDC）に感謝したいと試薬を提供するためのジョンズホプキンス大学公衆衛生学部の研究室です。

1。糞便サンプル<ol><li>糞便サンプルは、RNAを維持するために凍結保存する必要があります。 10パーセント糞便懸濁液を作成するには、解凍糞便サンプルの約0.1グラムをとり、PBS 1 mlに完了します。 </li><li>繰り返し凍結融解を避けるために、200μlのアリコート。 -70℃でアリコートを格納する</li><li>解凍および抽出に使用する前に、10分間4000グラムでアリコートを遠心分離します。 </li></ol> 2。グアニジン＆シリカの抽出のためのシリカ粒子の調製<ol><li> RTで、二酸化珪素の30グラムを中断し、最大250 mLのdH 2 Oで完了します。 </li><li>沈降の24時間後、吸引により上清215 mLを削除します。最大250 mLまでのdH 2 Oを追加し、振とうすることによりシリカペレットを中断します。 </li><li>別の24時間後、吸引により上清を除去し、塩酸（HCl）を用いて、pHを2.0にシリカ懸濁液のpHを調整します。アリコートガラスのボットで得られたシリカ懸濁液tles、オートクレーブ。 </li></ol> 3。グアニジン＆シリカの抽出のためのL6バッファーの調製<ol><li>室温で、0.1 Mトリス塩酸50mLのpHは6.4と0.2 M EDTA、pH8.0の溶液を11 mLのグアニジニウムチオシアネート（GuSCN）とトリトンX-100の1.3グラムの60グラムを溶解させたL6バッファを準備します。 </li></ol> 4。グアニジン＆シリカの抽出のためのL2バッファの準備<ol><li>室温で、0.1 Mトリス塩酸150mLを、pHを6.4にグアニジニウムチオシアネート（GuSCN）の180グラムを溶解させたL2バッファを準備します。 </li></ol> 5。抽出法<ol><li>各抽出年11肯定的な糞便サンプルは、ポジティブコントロール、およびDEPC処理水として、ネガティブコントロールとして、含まれるべきである。 </li><li>マイクロ遠心チューブに次のように混ぜる。バッファL6、シリカ粒子の20μLを1 mL、10％糞便抽出懸濁液200μLを追加します。簡単にボルテックスし、15マイルRTで残すN。 </li><li> 10秒間6000グラムで、懸濁液を遠心分離し、70％エタノールで2回洗浄し、アセトンで1時間、続いて二回バッファL2の1ミリリットルでペレットを洗浄する。 56℃乾燥した加熱ブロックにペレットを乾燥℃で5分間。 </li><li> RNaseフリーのdH 2 O50μLを追加することにより、シリカペレットの水和物は、RNA。 15分間RNasinの1μL、56で反転管とインキュベートすることにより混合°Cを追加します。 </li><li>卓上遠心機で、フルスピードで3分間遠心し、RNAを含む上清の40μLを収集します。 </li><li>光度計2000分光光度計（サーモフィッシャーサイエンティフィック）を使用して抽出したRNAサンプルを定量化する。次いで-70℃で保存を使用するまで、6ヶ月まで。 （ 注：完全なトータルRNAを維持する良い方法は、cDNAに変換されます）。 </li></ol> 6。商業RNA別途準備ウイルスRNAキットを使用して別の抽出完全な手順については、冊子プロブで発見されQIAGENキットでided。簡単に手順は次のとおりです。 <ol><li>バッファAVLのピペットで560μLを1.5 mLチューブにキャリアRNAを含む、10％糞便懸濁液140μLを追加します。 15秒間パルスボルテックスで混和。 10分間室温でインキュベートします。 </li><li> 15秒間パルスボルテックスサンプルと混合するエタノール56​​0μL（96〜100％）を追加し、手短に遠心します。 </li><li> 2 mlのコレクションチューブに入れ、スピンカラムに、このソリューションの630μLを適用します。 6000グラムで1分間遠心してから、クリーンな2 mLのチューブにスピンカラムを配置します。 </li><li> 1分間6000グラムで、バッファAW1および遠心の500μLと結合したRNAを含むカラムを洗浄します。きれいな2 mlのコレクションチューブにカラムを配置します。 </li><li> 3分間、フルスピード（20,000 gまたは14,000 rpmで）500 Buffer AW2をμLの遠心でカラムを洗浄します。 </li><li> 、新しい1.5 mlマイクロ遠心チューブにスピンカラムを置き40μLDEPC処理水を追加し、フルスピードでRNAを溶出します。を繰り返す溶出したRNAの最後の80μL用のce。 </li><li>光度計2000分光光度計（サーモフィッシャーサイエンティフィック）を使用して抽出したRNAサンプルを定量化する。次いで-70℃で保存を使用するまで、6ヶ月まで。 （ 注：完全なトータルRNAを維持する良い方法は、cDNAに変換されます）。 </li></ol> 7。特異的なTaqManプローブによるワンステップリアルタイムRT-PCRにより抽出したRNA中のノロウイルスの検出楽器StepOnePlusリアルタイムPCRシステム（Applied Biosystems社）。 方法論<ol><li>潜在的なRNaseのコンタミを除去するためにAWAY RNase Aですべての作業面、ピペット、および遠心分離機を拭いて清掃してください。 </li><li>ピペットNOV GIとGIIスクリーニングマスターは、 表1によるとミックスします。プライマーとTaqManプローブは、 表2に記載されています。 </li><li>各反応チューブに適したマスターミックスのアリコートを10μL。 </li><li>原液未知のサンプルの5μLを追加します。対応する反応ウェルにRNAを、陰性対照としてDEPC処理水、またはそれぞれGI GII陽性コントロールRNA、。すべての検体をduplicateで実行する必要があります。それぞれ300μg/μLのおよび500μg/μLの濃度の正のコントロールと基準は、疾病管理予防のための国立カリシウイルス研究所センター（CDC）によって提供されていました。 </li><li> 10秒のために6000グラムで、反応プレートを遠心します。 </li><li>実験のプロパティ画面で、実行のための実験のタイプを定義して選択します。のTaqMan試薬は試薬の種類、およびその前のPCR増幅読み取りと選択されているように表示されていることを確認します。 </li><li> 表3に示す温度プロファイルを用いて15μLの反応を実行します。 </li><li>結果を分析します。 </li></ol> 8。従来のRT-PCRおよびシーケンシングを用いたGIとGII月のジェノタイピング （それが一般的で広く使われている方法ですので、それはこのビデオに記載されていません。） 楽器。アプライドバイオシステムズStepOneとQuantitecテンプレートを使用してStepOnePlusリアルタイムPCRシステム。 方法論<ol><li>タイピングの前に、サンプルの遺伝子型（GIまたはGII）は、RT-PCRは、上記のスクリーニングによって決定されます。 GI NOV RNAは、GIのジェノタイピングマスターミックスとGII遺伝子タイピングマスターミックスとGII月RNAを増幅する必要があります。 </li><li>ピペットNOV GIとGII遺伝子タイピングマスターは表4に従って混合します。プライマーGI SKF / SKRとG2 SKF / SKRは、 表5に記載されています。 </li><li> 0.2mLの反応チューブに適したマスターミックスのアリコートを45μL。 </li><li>それぞれ陰性対照チューブにDEPC処理水の5μL、事前スクリーニング、11（GIおよび/またはGII）は、対応する反応チューブへの積極的なRNAの5μLを追加します。 </li><li> 表6の熱プロファイルを使用して50μLの反応を実行します。 </li><li> PCR産物contaiを送る少なくとも100は、増幅されたカプシド領域のng /μLの精製と配列決定のために会社をマクロゼンする寧。 （9700大セネカハイウェイ。ロックビル、MD 20850およびシークエンシングサンプルあたり10ドル）。 </li></ol> 9。代表的な結果<img src="/files/ftp_upload/3232/3232fig1.jpg" alt="図1" /> 図1は、TaqMan下痢子供から糞便サンプルから抽出したRNAアッセイに使用されるワンステップRT-PCRから代表的な結果を示しています。ポジティブサンプルの閾値サイクル（Ct）がGIIのGIとCt 39未満またはCT 37に等しく設定された。ポジティブコントロール用のCT-値は、それぞれ27およびGIとGIIのためのORF1-ORF2のジャンクションの18未満であることが判明した。 <a href="http://www.jove.com/files/ftp_upload/3232/3232fig1large.jpg" target="_blank">拡大図を表示するには、ここをクリックしてください</a> 。 <img src="/files/ftp_upload/3232/3232fig2.jpg" alt="図2" /> 図2 </strongは&gt;シリカ方法グアニジンとQiAmpウイルスRNAキットでのCt値の頭に頭の比較を示しています。両方のテストのデータは平等線（傾き= 1と切片= 0）に非常に近い分類されます。これは、回帰分析と相関係数によって確認されています。すべてのネガティブコントロールは、両方の方法で評価し、0であることが判明した。 <table border="1"><tbody><tr><td> マスターミックス </td><td> 最終濃度 </td><td> 体積（μL） </td></tr><tr><td> H 2 0 PCR </td><td></td><td> 2.875 </td></tr><tr><td>のQuantiTect RT-PCRマスターミックス</td><td> 1X </td><td> 6.250 </td></tr><tr><td> RTミックス</td><td> 1X </td><td> 0.125 </td></tr><tr><td> 50μMコグRプライマー</td><td> 1μM </td><td> 0.250 </td></tr><tr><td> 50μMコグFプライマー</td><td> 1μM </td><td> 0.250 </td></tr><tr><td> 10μMリングプローブ</td><td> 1μM </td><td> 0.125/0.250 * </td></tr><tr><td></td><td></td><td> 10.00 </td></tr></tbody></table>各プローブの0.125μLがGIIのテストに使用している間に、各プローブの* 0.250μLは、GIテストのために使用されていました。 スクリーニングGIとGIIについては、 表1を参照。キアゲンのQuantiTectマスターミックス（トルヒーヨら 、2006）7。 <table border="1"><tbody><tr><td> 名 </td><td> ジェノグループ </td><td> 使用 </td><td> 配列（5 &#39;から3&#39;） </td></tr><tr><td>コグ1R </td><td> GI </td><td>プライマー</td><td> CTT AGA CGC CAT CAT CAT TYA C </td></tr><tr><td>コグ1F </td><td> GI </td><td>プライマー</td><td> CGY TGG ATG CGN TTY CAT GA </td></tr><tr><td>コグ2R </td><td> GII </td><td>プライマー</td><td> TCG ACG CCA TCT TCA TTC ACA </td></tr><tr><td>コグ2F </td><td> GII </td><td>プライマー</td><td> CAR GAR BCN ATG TTY AGR TGG ATG AG </td></tr><tr><td>リング1A </td><td> GI </td><td>プローブ</td><td> FAM-AGA TYG CGA TCY CCT GTC CA-BHQ-1 </td></tr><tr><td>リング1B </td><td> GI </td><td>プローブ</td><td> FAM-AGA TCG CGG TCT CCT GTC CA-BHQ-1 </td></tr><tr><td> Ring2-TP </td><td> GII </td><td>プローブ</td><td> FAM-TGG GAG GGC GAT CGC AAT CT-BHQ-1 </td></tr></tbody></table> genogroups I、II（影山ら 、のリアルタイム定量的RT-PCRに使用される表2のプライマーおよびプローブオリゴヌクレオチド2003）8。 <table border="1"><tbody><tr><td> 手順 </td><td> 温度（°C） </td><td> 時間（分） </td><td></td></tr><tr><td> 1 </td><td> 50 </td><td> 30:00 </td><td> cDNA合成</td></tr><tr><td> 2 </td><td> 95 </td><td>午前15時</td><td> HotStartのTaqポリメラーゼの活性化</td></tr><tr><td> 3 </td><td> 95 </td><td>夜12時15分</td><td></td></tr><tr><td></td><td> 60 </td><td> 1時</td><td> 45Xサイクル</td></tr></tbody></table> テーブル（3）ワンステップのTaqManリアルタイムRT-PCR用のサーマル·プロファイル（トルヒーヨら 、2006）7。 <table border="1"><tbody><tr><td> マスターミックス </td><td> 最終濃度 </td><td> 体積（μL） </td></tr><tr><td> H 2 0 PCR </td><td></td><td> 29.50 </td></tr><tr><td> 5XキアゲンRT-PCRバッファー</td><td> 1X </td><td> 10.00 </td></tr><tr><td> 10mMのdNTPミックス</td><td> 0.4mMの</td><td> 2.00 </td></tr><tr><td>酵素ミックス</td><td></td><td> 2.00 </td></tr><tr><td> 40 U /μLRNAsin </td><td> 20 U /μL </td><td> 0.50 </td></tr><tr><td> 10μMSKF </td><td> 0.1μM </td><td> 0.50 </td></tr><tr><td> 10μMのSKR </td><td> 0.1μM </td><td> 0.50 </td></tr></tbody><tbody><tr><td></td><td></td><td> 45.00 </td></tr></tbody></table> 表4。キアゲン遺伝子型GIとGIIのためのワンステップRT-PCRマスターミックス。 <table border="1"><tbody><tr><td> 名 </td><td> ジェノグループ </td><td> 使用 </td><td> 配列（5 &#39;から3&#39;） </td></tr><tr><td> G1SKF </td><td> GI </td><td>プライマー</td><td> 5&#39;-CTG C​​CC GAA TTY GTA AAT GA - 3 </td></tr><tr><td> G1SKR </td><td> GI </td><td>プライマー</td><td> 5&#39;-CCA ACC CAR CCA TTR TAC A - &#39;3 </td></tr><tr><td> G2SKF </td><td> GII </td><td>プライマー</td><td> 5&#39;-CNT GGG AGG GCG ATC GCA A - 3 </td></tr><tr><td> G2SKR</td><td> GII </td><td>プライマー</td><td> 5&#39;-CCR CCN GCA TRH CCR TTR TA CAT-3 </td></tr></tbody></table> 表5。キャプシド領域の領域C（小島ら 、2002）の増幅に用いるプライマー5。 <table border="1"><tbody><tr><td> 手順 </td><td> 温度（°C） </td><td> 時間（分） </td><td></td></tr><tr><td> 1 </td><td> 60 </td><td> 30:00 </td><td> cDNA合成</td></tr><tr><td> 2 </td><td> 96 </td><td>午前15時</td><td> HotStartのTaqポリメラーゼの活性化</td></tr><tr><td> 3 </td><td> 94 </td><td>夜12時03分0 </td><td></td></tr><tr><td></td><td> 52 </td><td> 1時</td><td> 40Xサイクル</td></tr><tr><td></td><td> 72 </td><td>夜12時30 </td><td></td></tr><tr><td> 4 </td><td> 72 </td><td> 10時</td><td>アニーリング</td></tr></tbody></table> 表6のTaqManワンステップRT-PCR用のサーマル·プロファイル。

<ol>
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糞便サンプルから核酸を単離するための経済的なインハウス方式を使用して、我々は、キアゲンから商用QIAmpウイルスRNAキットと同じように同じ結果を取得し、一緒に私たちの研究室で開発されたTaqMan RT-PCRで、私たちは月の広い範囲を検出することができますGIとGII遺伝子型に属する品種。領域Cプロトコルの最近の出版物は78％6のタイピング速度を報告した。現代のNOV株の多様性が?...

<table border="1"><tbody><tr><td> 試薬の名前 </td><td> 会社 </td><td> カタログ番号 </td><td> コメント </td></tr><tr><td>グアニジンイソチオシアネート</td><td>シグマAdrich </td><td> G9277 </td><td></td></tr><tr><td>トリスHCl </td><td> Sigma-Aldrich社</td><td> T5941 </td><td></td></tr><tr><td> EDTA </td><td> Sigma-Aldrich社</td><td> E5134 </td><td></td></tr><tr><td>シリカ</td><td> Sigma-Aldrich社</td><td> S5631 </td><td></td></tr><tr><td>トリトンX 100 </td><td> BDHケミカル</td><td> 14530 </td><td></td></tr><tr><td>ジエチル</td><td> Sigma-Aldrich社</td><td> D-5758 </td><td></td></tr><tr><td>別途準備ウイルスRNA Miniキット（250） </td><td> QIAGEN </td><td> 52906 </td><td></td></tr><tr><td>定量テックプローブRT-PCRキット（200） </td><td> QIAGEN </td><td> 204443 </td><td></td></tr><tr><td>キアゲンワンステップRT-PCRキット（200） </td><td> QIAGEN </td><td> 210212 </td><td></td></tr><tr><td> RNase阻害剤2000個</td><td> A.Biosystems </td><td> N808-0119 </td><td> 2000 UNIDが/バイアル</td></tr><tr><td>ノンスティックRnaeフリーマイクロチューブ</td><td> Ambionの</td><td> AM12450 </td><td></td></tr><tr><td>純アガロース1000年</td><td>インビトロジェン</td><td> 16550-100 </td><td></td></tr></tbody></table>

detection and genogrouping of noroviruses from children's stools by taqman one-step rt-pcr

ノロウイルス（NoVs）はすべての年齢の人間の世界的な散発的な急性胃腸炎の集団発生の主要原因となっています。彼らは、ロタウイルスに似た公衆衛生上の影響を持つ子どもの入院の重要な原因となっています。 NoVsは偉大な遺伝的多様性のRNAウイルスであり、新しい株の連続的な外観があります。五genogroupsが認識され、GIとGIIを彼らの多くの遺伝子型とヒトへの感染のために最も重要なサブタイプである。しかし、これら2つの遺伝子型の診断は、診断と治療を遅らせることは、問題のままである。1、2、3 糞便検体からのRNA抽出のために最も一般的に使用されるメソッドは、QiagenからQIAmpウイルスRNA市販のキットです。このメソッドは、コントロールRNaseがそのバッファを、シリカゲルメンブレンの結合特性を組み合わせて、マイクロスピンの速度と一緒にカラムにRNAの最適な結合を提供しています。このメソッドは、簡単で高速かつ信頼性が高く、カーです。製造元から提供されている記述で詳述されているいくつかのステップでiedを。 ノロウイルスは、下痢の最も一般的な原因として、ロタウイルスに次ぐです。ノロウイルスの診断は、下痢の病因のすべての研究で同様の発生、または個々の下痢症で使用できる必要があります。現在ではしかし、ノロウイルスの診断は、診断の簡単な方法の欠如に起因するわずか数センターに限定されています。この遅延の診断と治療1、2、3。さらに、国内および間に腐食性のバッファのコストと規制輸送に起因するこれらの製造キットの使用ロジスティック問題を引き起こします。結果として、このプロトコルで我々は、グアニジニウムチオシアネートの抗ヌクレアーゼのプロパティと一緒にシリカ粒子の核酸結合特性を使用して、元のブームらの方法4に基づいて、代替的、経済的、社内の方法を説明。 <p class=&quot;jove_content&quot;&gt;糞便検体からNoVs分離株の検出とgenogrouping（GIとGII）は、異なるターゲットを利用したいくつかのRT-PCRのプロトコルが開発されている。コンセンサスは、高感度、特異性、高スループットと使いやすさと再現性を兼ね備えているためのTaqManケミストリーを用いたRT-PCRは、診断のための最良の分子技術であろうということである。診断に最も適したのでNOVのゲノムの中で最も保存領域と、ここではオープンリーディングフレーム1（ORF1）-ORF2のジャンクショ​​ン領域を標的とするアッセイを説明します。さらに、遺伝子解析カプシド（領域C）もともと小島らによって記載されたプライマーを用いて5の主要タンパク質から高可変N末端 ​​シェルをターゲットにして、従来のRT-PCRのために詳述されている。従来のPCRからのPCR産物のシークエンシングは、GIとGII genogroupsに属する遺伝子の分化を可能にします。

Watch this Scientific Journal Video about Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR at JoVE.com

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

オープンリーディングフレーム1（ORF1）ORF2接合領域に特異的なプライマーとTaqManプローブを利用して子どもの便からノロウイルス分離株の検出および遺伝子型の識別のためのワンステップRT-PCRアッセイ、ノロウイルスゲノムの中で最も保存領域です。説明します。非商業的、費用対効果の高いRNA抽出法が詳述されている。

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 ...

detection-genogrouping-noroviruses-from-children-s-stools-taqman-one

Research

JoVE Journal

Medicine

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

17.2K ビュー.  Universidad Peruana Cayetano Heredia. オープンリーディングフレーム1（ORF1）ORF2接合領域に特異的なプライマーとTaqManプローブを利用して子どもの便からノロウイルス分離株の検出および遺伝子型の識別のためのワンステップRT-PCRアッセイ、ノロウイルスゲノムの中で最も保存領域です。説明します。非商業的、費用対効果の高いRNA抽出法が詳述されている。

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

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

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

がん治療のモニタリングのためのチェレンコフ発光イメージング（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

マウスモデルにおける下大静脈介在移植の移植

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

病気のモデリングのための筋生検からの患者由来の細胞株の単離と不死

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

全組織生検からヒト上皮EnteroidsとColonoidsの確立

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

膵管注入:薬物およびウイルス送達の有効かつ選択的な方法

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

非放射性 L azidohomoalanine ラベル法による新生タンパク質合成解析用プライマリ マウス肝細胞の分離

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

ハンドヘルド金属探知器検診小児における金属異物摂取

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

VASP のリン酸化を測定することによって血液中の一酸化窒素の血小板ベースの検出

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Assessment and Characterization of Hyaloid Vessels in Mice

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed regression of hyaloid vessels can be seen in diseases such as persistent hyperplastic primary vitreous (PHPV), leading to an obstructed light path and impaired visual function. Understanding the mechanisms underlying the hyaloid vessel regression may lead to new molecular insights into the vascular regression process and potential new ways to manage diseases with persistent hyaloid vessels. Here we describe the procedures for imaging hyaloid in live mice with optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and a detailed technical protocol of isolating and flat-mounting hyaloid ex vivo for quantitative analysis. Low-density lipoprotein receptor-related protein 5 (LRP5) knockout mice were used as an experimental model of persistent hyaloid vessels, to illustrate the techniques. Together, these techniques may facilitate a thorough assessment of hyaloid vessels as an experimental model of vascular regression and studies on the mechanism of persistent hyaloid vessels.

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

マウスにおけるヒアロイド血管の評価と特徴付け

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...