This study was supported by a K08 Award (DK090154-01) from the National Institutes of Health (NIH; to F.M.S.).

内視鏡的逆行性胆道膵管造影（ERCP）によって患者から胆汁を取得する治験審査委員会によるヒト被験者の治験実施計画書の承認を必要とします。ここで提示すべての作業は、ジョンズ・ホプキンス大学の治験審査委員会によって承認されました。 胆汁から1.エキソソームの単離<ol><li>内視鏡的逆行性胆道膵管造影（ERCP）を実行します。仰臥位で患者を置き、挿管。 <ol><li>患者の口を通って十二指腸を通過し、十二指腸の第二部に挿入し、主要な乳頭を識別します。 </li><li>選択トリプルルーメンは0.035インチ（0.9ミリメートル）親水性ガイドワイヤが事前にロードされた括約筋切開刀挿入することにより、総胆管にカニューレを挿入。 </li><li>機会に、右肝管から胆嚢管を区別することが困難な場合があるため、左肝管に透視下でガイドワイヤを進め、次に進めます安定した位置を取得するための胆管への括約筋切開刀5センチメートル。 </li><li>ルアーロックを介してガイドワイヤポートに10ミリリットル注射器を取り付け、ガイドワイヤを外し、負圧の10ミリリットルを使用して胆汁を吸引します。 </li><li>ガイドワイヤーを再挿入し、胆管系を不透明化するために注入口を介して造影剤を注入することにより、ERCPを続行するために胆汁を含む注射器を取り外します。 </li><li>氷上で15ミリリットルチューブに回収胆汁を移し、遠心分離ステップを続行する準備ができるまで氷上に保ちます。 注意：自分自身とサンプルを保護するために手袋を着用してください！それはA型肝炎ウイルス粒子を含有することができるように感染の潜在的な供給源として胆汁トリート！ </li><li>氷上でのサンプル（複数可）してください。いずれかの10分間、4℃で500×gでステップ1.2または遠心分離機を続行し、さらに使用するまで-80℃で凍結します。 注：数年前からこの方法で保存された試料は、日常的に良い結果19で使用されています。 </li></ol&gt; </li><li>微量遠心管に胆汁の400μLを移し、4℃で10分間、300×gでの遠心分離により細胞および細胞破片を集めます。 </li><li>ピペットで上清を除去し、さらに破片の上清をクリアするために4℃でさらに20分間、16,500×gで新鮮なマイクロ遠心チューブと遠心分離機にサンプル（複数可）を転送します。バイオハザード廃棄物にチューブを捨てます。あるいは、代わりに、超遠心機でサンプルをスピン。 </li><li>上清を収集し、左から200nmよりも大きい任意の粒子を除去するために結合の良好な流量と低タンパク質を持っている0.2μmのポリエーテルスルホン（PES）膜フィルターを通してシリンジで生物学的安全キャビネットの中でそれをフィルタリングします。 </li><li>ピペット管を超遠心し、チューブが完全な2/3以上になるようにPBSを追加するために濾過した上清。チューブはそれより少ない液体が含まれている場合、チューブが遠心分離中に崩壊します！チューブは、洗浄オートクレーブと数回再使用することができます。 </li><li>ペルら4℃で70分間120,000×gで超遠心分離を通じてエキソソーム。時々小さな黄色のペレットを参照するには、超遠心分離管の底部での遠心分離を見た後、これらはエキソソームです。 </li><li>慎重にデカントすることによって上清を捨て、10％v / vの最終濃度に漂白剤を添加することにより、廃棄物を消毒し、20分間放置。 </li><li>下流の実験のために、いずれかの適切な溶液の小体積のペレット（複数可）を処理（ すなわち、放射性免疫沈降アッセイ（RIPA）タンパク質の単離、のmiRの単離のためのRNA溶解緩衝液のための緩衝液）またはリン酸の50〜150μl中に再懸濁バッファリング生理食塩水、将来の使用のために-80℃で保存することができるいずれか（PBS）（定量的リアルタイムポリメラーゼ連鎖反応（QRT-PCR）またはタンパク質の抽出など ）またはエキソソームTEMによって調製またはナノ粒子の光学分析の特徴付けのために使用さ。 </li></ol> 2.エキソソーム同定透過型電子顕微鏡（TEM）またはCryoEM <ol><li>ピペッティングにより100μlのPBSで細胞外小胞を再懸濁します。初めての分離を行う場合は特に理想的には、-20℃で保存するか、またはされていません-80℃〜エキソソームを使用します。 </li><li> 2分間の400メッシュの炭素被覆Parlodionの銅グリッドに20μlのアリコート（約2μgの）を吸着し、室温で乾燥させます。 </li><li>乾燥製剤に慎重に滴を配置することにより、室温で5分間、1％（v / v）のグルタルアルデヒド（EMグレード）にエキソソームを修正しました。 </li><li> 5分間、水で2回グリッドを洗浄した後、30秒間、1％リンタングステン酸（PTA）で染色電気自動車を対比。 </li><li> 20,000の倍率で開始し、粒子の大きさを決定する際100,000に増加80キロボルトの加速電圧で電子顕微鏡で画像を取得します。 注：ホルムバール炭素でコーティングされた200メッシュの銅やニッケルグリッドと対比染色李上に重ね着のような他のプレップ10~15分間KE 1％または2％酢酸ウラニルも可能です。 </li><li> （：3比1）CryoEMのために、60μlのプロテ​​インGの金を10nmと20μlのアリコート（約2μgの）を混ぜます。これは、エキソソームの直径を決定するのに役立ちます。 </li><li>グロー放電さC-フラットホーリーカーボングリッド上にサンプルを転送し、ブロッティングにより、過剰な液体を除去します。直ちに液体エタンにそれらを浸漬することにより、グリッドをフリーズします。 </li><li>液体窒素中cryoholderにグリッドをマウントします。 </li><li> 20,000と100,000の倍率で200kVで画像を取得します。 </li></ol>マルチパラメータナノ粒子の光学分析による3エキソソーム同定<ol><li>ピペッティングにより100μlのPBSで孤立エキソソームを再懸濁します。 </li><li>いくつかの異なる比で600μlのPBS中のエキソソームを希釈（のような1時50分、1：100、1：300、1：600および1：1200）。 </li><li> manufactuに応じてリアルタイムで適切な機器およびソフトウェアを用いてレーザー光散乱によってエキソソームを可視化RERのプロトコル20。 </li><li>粒子が見えるようになるまで、粒子が滑らかに見えるようにフォーカスを調整し、撮影モードでカメラのレベルを上げます。 </li><li>標準モードでは、粒子はまだ見ることができる最低レベルにカメラを減少しながら、必要なカメラのシャッターとゲインを調整する場合は、これを達成します。 </li><li>視覚ユニットをフラッシュし、PBSで希釈調整しない場合は、20から100の粒子が画面に表示されていることを確認して下さい。 </li><li>その後、30-60間の秒にキャプチャ持続時間を調整します。ビデオがキャプチャされた後、全ての粒子が画面上で確認されるまでの処理パラメータを調整し、ビデオファイルを処理します。 </li></ol>ウエスタンブロット4.エキソソーム特性評価<ol><li>氷冷放射性免疫沈降アッセイ（RIPA）の100μl中の胆汁2-5 mlで溶解エキソソームペレットピペッティングによりプロテアーゼ阻害剤でバッファリングし、ダウン徹底的に30秒間激しく溶解物を混ぜます効果的にタンパク質を可溶化する渦に。 </li><li>ほとんどの時間必要ではないが、完全な溶解を確実にするために10秒ごとに、2パルスと氷上でサンプルのパルス超音波処理を使用します。 </li><li> 5分間、4℃で15,000×gで遠心分離することによって不溶性物質をペレット化し、清潔なチューブに上清を移します。 </li><li>選択の方法、製造業者のプロトコルに従って（ すなわち 、ビシンコニン酸（BCA）、クマシー、修正ローリー、660 nmのタンパク質アッセイなど ）でタンパク質濃度を決定します。同様の方法は、実験間で比較することができる結果を達成するために一貫して使用される方法は、長いほど重要ではありません。 </li><li>電気泳動用ポリアクリルアミドゲル（PAGE）のウェルに5分間95℃で試料を加熱した後、負荷Laemmli緩衝液中でサンプル当たり50μgのタンパク質。 </li><li> 1.5時間PAGEゲル上でサンプルを電気泳動し、ナイロン膜上に分離されたタンパク質を移します。 </li><li> B次いで、希釈した抗CD63及び抗TSG-101のようなエキソソーム特異的抗体を用いてインキュベートし、1時間、0.1％のTween 20（TBS-T）を用いてトリス緩衝生理食塩水中の5％脱脂乳で膜（複数可）をロックします1の：500 4℃で、好ましくは、O / N。 </li><li> TBS-T 3×10分を有する膜を洗浄し、その後、室温で1-2時間、TBS-T中の5％脱脂乳にHRP結合二次抗体と一致する、適切でインキュベートします。 </li><li>膜（複数可）TBS-Tで3×5分間洗浄し、製造業者のプロトコルに従って、化学発光試薬で特定のタンパク質を検出します。 </li><li>イメージフィルムか、製造元のプロトコルに従ってデジタルイメージングシステムを有する膜。 </li></ol>単離されたエキソソームから5マイクロRNAの単離注：胆汁エキソソームからmiRNAの単離は、市販のキットに基づいて修正されたmiRNAの単離を用いて行われるが、他の任意のRNA単離方法を用いるまたは適合させることができます。 <ol><li> 300＆を追加＃181;リットル（400μlの胆汁から単離された）エキソソームペレットに溶解/ビル・バッファは、渦、ピペットまたは完全に均一溶解液を得るためにエキソソームを溶解するために、針/注射器を通過します。 </li><li> miRNAのホモジネート添加剤の1月10日（30μL）ボリュームを追加し、ボルテックスまたはチューブを数時間を反転させてよく混ぜます。 </li><li>氷上で10分間ミックスをインキュベートします。 </li><li>酸性フェノールのボリュームを追加します。最初のホモジネート（300μl）を等しいクロロホルムを。 </li><li> 5フェムトモル/μlのCEL-のmiR 39および30から60秒間渦の5μlを添加します。 </li><li>水（上）と有機（下）の相を分離するために、室温で万×gで5分間遠心分離します。 </li><li>転送量に注意しながら、慎重に新しいチューブにピペッティングし、転送することにより、上相を吸引します。適切な有機廃棄物容器内の有機相を捨てます。 </li><li>低分子RNAを濃縮するためには、回収された水相に100％エタノールの1/3量を追加し、徹底的にTUをボルテックスまたは転倒混和します数回です。 </li><li>一度に700μlのまでピペットフィルターカートリッジへのライセート/エタノール混合物、。 </li><li>万XGの最大で15秒間のカートリッジを遠心分離やフィルターを通して混合物を渡すために真空を適用します。 </li><li>ろ液を収集し、溶解液/エタノール混合物は700μLを超えた場合、新しいチューブにフロースルーを転送し、すべてのろ液を収集するための手順を繰り返します。これらのフィルタは、低分子RNAを欠くRNA画分を含み、これらのより大きなRNAを回収するために使用することができます。 </li><li>総ろ液にRT 100％エタノールの2/3量を加え、ボルテックスで混和します。 </li><li>ピペット第二のフィルタカートリッジにろ液/エタノール混合物。一度に印加できる最大容量は700μlである前にのように。 </li><li>万×gで15秒間遠心分離またはステップ5.10で説明したように真空を適用します。 </li><li>フロースルーを捨て、ろ液/エタノール混合物のすべてがフィルタリングされるまで繰り返します。 </li><li> 700を適用します81;フィルターカートリッジへリットルのmiRNAの洗浄液1、5〜10秒間遠心分離または真空を適用し、フロースルーを捨てます。 </li><li>ステップ5.16で行われるように500μlの洗浄溶液2/3でフィルターを洗ってください。 </li><li> 、ステップ5.16のように洗浄溶液2/3の500μlの二回目の洗浄フロースルーを廃棄し、バックコレクションチューブにフィルターを置き繰り返します。 </li><li>フィルタからすべての残留流体を除去するために、万×gで1分間フィルターカートリッジをスピン。 </li><li>新鮮なコレクションチューブにフィルターカートリッジを移し、フィルタの中心に熱い（95℃）ヌクレアーゼを含まない水100μlを適用します。 </li><li> RNAを回収するために最大速度で20〜30秒間、アセンブリをスピン。 </li><li>溶出液を収集し、直ちに使用するか、または-80℃で保存します。 </li></ol>

<ol>
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Anticancer Ther. 11 (9), 1341-1343 (2011).</li><li>Li, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+bile+contains+microRNA-laden+extracellular+vesicles+that+can+be+used+for+cholangiocarcinoma+diagnosis.">Human bile contains microRNA-laden extracellular vesicles that can be used for cholangiocarcinoma diagnosis.</a> Hepatology. 60 (3), 896-907 (2014).</li><li>Gabriel, D. A., Giordano, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microparticle+sizing+and+counting+using+light+scattering+methods.">Microparticle sizing and counting using light scattering methods.</a> Semin. Thromb. Hemost. 36 (8), 824-832 (2010).</li><li>Colombo, M., Raposo, G., Thery, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biogenesis,+secretion,+and+intercellular+interactions+of+exosomes+and+other+extracellular+vesicles.">Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles.</a> Annu. Rev. Cell Dev. Biol. 30, 255-289 (2014).</li></ol>

None of the authors have competing financial interests.

確実胆汁から単離したエキソソームを利用するためには、引き換えに、高品質のサンプルを得るために、一貫した高品質の単離方法を用いることが重要です。本論文で定義された方法論は、ヒト胆汁からエキソソームとのmiRNAを単離するための十分に確立された方法です。これは、最低でも電子顕微鏡やナノ粒子の光学分析とウェスタンブロットを含むべきである単離されたエキソソームの?...

他の生物学的流体、すなわち、母乳、血漿または尿のように、胆汁はエキソソーム、脂質の豊富な小胞1-4が含まれています 。エキソソームは、誘導または受容細胞5、6、彼らの正常な機能4、7-9の一部である可能性があります細胞間コミュニケーションの形で生物学的機能を変化させることができます。エキソソームは、診断10のためのバイオマーカーの貴重な情報源を提供することができmiRNA種を含むことができます。 miRNAプロファイルは、疾患11-14の様々な診断および予後マーカーとして、近年かなりの注目を集めています。 miRNA自体は、おそらく死細胞により放出され、脱落細胞の量が大幅に基礎疾患によって影響を受けることができ、生物学的流体中に見出すことができます。エキソソームのmiRNAプロファイルは、必ずしも元のセル6、10、15から17のmiRNAプロファイルを反映していない、まだエキソソームは、親CEの特徴であるかもしれないのmiRNAの署名を運ぶことができます腫瘍細胞が好きでしょう。腫瘍由来エキソソームは、肺腺癌、前立腺癌および他の腫瘍8、16、18の患者の血漿中で同定されています。 この方法の目的は、彼らの前処理手順の大部分は独立したヒト胆汁標本からのエキソソームの信頼性の高い堅牢な単離であります。このような胆管19と胆管の疾患の潜在的な診断のためのmiRNAの信頼できる情報源としての胆汁を利用するために開発されました。これは、エキソソームを単離するために、速度の増加に伴って、遠心分離のいくつかのステップで構成され、他の生物学的流体に適用できる可能性があります。

<table><tbody><tr><td>0.2 &amp;micro;m Polyethersulfone (PES) membrane filter</td><td>Corning</td><td>431229</td><td></td></tr><tr><td>thinwall polyallomer ultracentrifuge tubes&amp;nbsp;</td><td>Beckman Coulter</td><td>331374</td><td></td></tr><tr><td>SW40Ti rotor</td><td>Beckman Coulter</td><td></td><td></td></tr><tr><td>LM10-HS&amp;nbsp;</td><td>Nanosight</td><td></td><td></td></tr><tr><td>Nanoparticle Tracking Analysis (NTA) software&amp;nbsp;</td><td>Nanosight</td><td></td><td></td></tr><tr><td>mirVANA</td><td>Life Technologies</td><td>AM1560</td><td></td></tr><tr><td>Complete Protease Inhibitor</td><td>Roche distributed by Sigma-Aldrich</td><td>4693116001</td><td></td></tr><tr><td>Immobilon PSQ</td><td>Millipore, Bedford MA</td><td>ISEQ00010</td><td></td></tr><tr><td>Anti-CD63 antibody</td><td>Abcam</td><td>ab59479</td><td></td></tr><tr><td>Anti-Tsg101</td><td>Abcam</td><td>ab30871</td><td></td></tr></tbody></table>

isolation and profiling of microrna-containing exosomes from human bile

最後の3年間のエキソソームの研究は大幅にバイオマーカーとその治療用途の同定および特徴付けに向けて範囲を拡張しました。 エキソソームは、最近、マイクロRNA（のmiR）を含有することが示されています。 MIRS自体は、診断目的のための貴重なバイオマーカーとして生じています。診療所や病院での検体採取が非常に可変であるように、全体の胆汁からのmiRNA分離は、実質的に変化します。簡単な方法で収集され胆汁サンプルから、ロバスト正確で再現性のmiRNAプロファイルを達成するために胆汁からエキソソームを単離し、特徴づけるために、高品質のプロトコルの開発を必要としました。方法は、いくつかの遠心分離および単離されたエキソソームをペレット化し、最終的な超遠心分離ステップで濾過工程を必要とします。電子顕微鏡、ウェスタンブロット、フローサイトメトリーおよびマルチパラメータナノ粒子の光学分析、電子の品質を検証するための重要な特性評価ステップである利用可能xosomes。これらのエキソソームからのmiRNAの単離のために、 線虫（Caenorhabditis elegans）のような種から非特異的、合成miRNAで溶解液をスパイク、 すなわち 、CEL-のmiR-39は、RNA抽出効率の正規化のために重要です。この方法以下の胆汁液からのエキソソームの単離に成功したmiRNAは-80℃で数年間保存胆汁サンプルからプロファイリングできます。

エキソソームは、通常の顕微鏡検査によって検出またはフローサイトメトリーするには小さすぎるので、電子顕微鏡またはナノ粒子の光学分析を行う必要があります。ナノ粒子の光学分析は、それはまた、定量的であり、サイズ分布および濃度を提供する電子顕微鏡に勝る利点を有します。計器は、サンプルにガラスプリズムを介して細く絞ったレーザビームを導入?...

Watch this Scientific Journal Video about Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile at JoVE.com

Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

ヒト胆汁からマイクロRNA含有エキソソームの単離とプロファイリング

Exosome research in the last three years has greatly extended the scope towards identification and characterization of biomarkers and their therapeutic ...

isolation-profiling-microrna-containing-exosomes-from-human

Universidad de Cartagena UDEC

Research

JoVE Journal

Medicine

10.2K ビュー.  Johns Hopkins University School of Medicine. Bile fluid is a valuable source of extracellular vesicles/exosomes that contain potentially important biomarkers. This protocol represents a robust method to isolate exosomes from human bile for further analyses including miRNA profiling.

ビデオ: Isolation and Profiling of MicroRNA-containing Exosomes from Human Bile

Isolation of High-density Lipoproteins for Non-coding Small RNA Quantification

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most interestingly, sRNAs are also found outside of cells and are stably present in all biological fluids. As such, extracellular sRNAs represent a novel class of disease biomarkers and are likely involved in cell signaling and intercellular communication networks. To assess their potential as biomarkers, sRNAs can be quantified in plasma, urine, and other fluids. Nevertheless, to fully understand the impact of extracellular sRNAs as endocrine signals, it is important to determine which carriers are transporting and protecting them in biological fluids (e.g., plasma), which cells and tissues contribute to extracellular sRNA pools, and cells and tissues capable of accepting and utilizing extracellular sRNA. To accomplish these goals, it is critical to isolate highly pure populations of extracellular carriers for sRNA profiling and quantification. We have previously demonstrated that lipoproteins, particularly high-density lipoproteins (HDL), transport functional microRNAs (miRNA) between cells and HDL-miRNAs are significantly altered in disease. Here, we detail a new protocol that utilizes tandem HDL isolation with density-gradient ultracentrifugation (DGUC) and fast-protein-liquid chromatography (FPLC) to obtain highly pure HDL for downstream profiling and quantification of all sRNAs, including miRNAs, using both high-throughput sequencing and real-time PCR approaches. This protocol will be a valuable resource for the investigation of sRNAs on HDL.

This protocol describes the isolation and quantification of high-density lipoprotein small RNAs.

非コード小型RNA定量化のための高密度リポタンパク質の単離

The diversity of small non-coding RNAs (sRNA) is rapidly expanding and their roles in biological processes, including gene regulation, are emerging. Most ...

生化学

Protein Digestion, Ultrafiltration, and Size Exclusion Chromatography to Optimize the Isolation of Exosomes from Human Blood Plasma and Serum

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and RNAs, are unique to the cells from which they are derived and can be used as indicators of disease. Several common enrichment protocols, including ultracentrifugation, yield exosomes laden with soluble protein contaminants. Specifically, we have found that the most abundant proteins within blood often co-purify with exosomes and can confound downstream proteomic studies, thwarting the identification of low abundance biomarker candidates. Of additional concern is irreproducibility of exosome protein quantification due to inconsistent representation of non-exosomal protein levels. The protocol detailed here was developed to remove non-exosomal proteins that co-purify along with exosomes, adding rigor to the exosome purification process. Five methods were compared using paired blood plasma and serum from five donors. Analysis using nanoparticle tracking analysis and micro bicinchoninic acid protein assay revealed that a combined protocol utilizing ultrafiltration and size exclusion chromatography yielded the optimal vesicle enrichment and soluble protein removal. Western blotting was used to verify that the expected abundant blood proteins, including albumin and apolipoproteins, were depleted.

Here, we present a protocol to purify exosomes from both plasma and serum with reduced co-purification of non-exosomal blood proteins. The optimized protocol includes ultrafiltration, protease treatment, and size exclusion chromatography. Enhanced purification of exosomes benefits downstream analyses, including more accurate quantification of vesicles and proteomic characterization.

エクソソームおよび人間の血液からの分離を最適化するためにタンパク質の消化が、限外濾過、サイズ排除クロマトグラフィー

Exosomes, a type of nanovesicle released from all cell types, can be isolated from any bodily fluid. The contents of exosomes, including proteins and ...

Preparation of Exosomes for siRNA Delivery to Cancer Cells

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, and intrinsic capability in intercellular delivery of various biomolecules. This work establishes an isolation protocol to achieve high yield and high purity of exosomes for siRNA delivery. Human Embryonic Kidney cells (HEK-293 cells) are cultured in bioreactor flasks and the culture supernatant (hereon referred to as conditioned medium) is harvested on a weekly basis to allow for enrichment of HEK-293 exosomes. The conditioned medium (CM) is pre-cleared of dead cells and cellular debris by differential centrifugation and is subjected to ultracentrifugation onto a sucrose cushion followed by a washing step, to collect the exosomes. Isolated HEK-293 exosomes are characterized for yield, morphology and exosomal marker expression by nanoparticle tracking analysis, protein quantification, electron microscopy and flow cytometry, respectively. Small interfering RNA (siRNA), fluorescently labeled with Atto655, is loaded into exosomes by electroporation and excess siRNA is removed by gel filtration. Cell uptake in PANC-1 cancer cells, after 24 h incubation at 37 &#176;C, is confirmed by flow cytometry. HEK-293 exosomes are 107.0 &#177; 8.2 nm in diameter. The exosome yield and particle-to-protein ratio (P:P) ratio are 6.99 &#177; 0.22 &#215; 1012 particle/mL and 8.3 &#177; 1.7 &#215; 1010 particle/&#181;g, respectively. The encapsulation efficiency of siRNA in exosomes is ~ 10-20%. Forty percent of the cells show positive signals for Atto655 at 24 h post-incubation. In conclusion, exosome isolation by ultracentrifugation onto sucrose cushion offers a combination of good yield and purity. siRNA could be successfully loaded into exosomes by electroporation and subsequently delivered into cancer cells in vitro. This protocol offers a standard procedure for developing siRNA-loaded exosomes for efficient delivery to cancer cells.

An exosome is a new generation of drug delivery carriers. We established an exosome isolation protocol with high yield and purity for siRNA delivery. We also encapsulated fluorescently labelled non-specific siRNA into exosomes and investigated the cellular uptake of siRNA-loaded exosomes in cancer cells.

がん細胞への siRNA のエクソソームの準備

Extracellular vesicles, in particular exosomes, have recently gained interest as novel drug delivery vectors due to their biological origin, abundance, ...

がん研究

Isolating, Sequencing and Analyzing Extracellular MicroRNAs from Human Mesenchymal Stem Cells

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, milk and urine. One subset of these RNAs are the posttranscriptional regulators &#8211; microRNAs (miRNAs). To delineate the miRNAs produced by specific cell types, in vitro culture systems can be used to harvest and profile exRNAs derived from one subset of cells. The secreted factors of mesenchymal stem cells are implicated in alleviating numerous diseases and is used as the in vitro model system here. This paper describes the process of collection, purification of small RNA and library generation to sequence extracellular miRNAs. ExRNAs from culture media differ from cellular RNA by being low RNA input samples, which calls for optimized procedures. This protocol provides a comprehensive guide to small exRNA sequencing from culture media, showing quality control checkpoints at each step during exRNA purification and sequencing.

This protocol demonstrates how to purify extracellular microRNAs from cell culture media for small RNA library construction and next generation sequencing. Various quality control checkpoints are described to allow readers to understand what to expect when working with low input samples like exRNAs.

分離、シーケンスおよびヒト間葉系幹細胞からの細胞のマイクロ Rna を分析

Extracellular and circulating RNAs (exRNA) are produced by many cell types of the body and exist in numerous bodily fluids such as saliva, plasma, serum, ...

遺伝学

Isolation of Exosomes from the Plasma of HIV-1 Positive Individuals

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. They are found in a number of biological fluids and are known to carry a variety of proteins, lipids, and nucleic acid molecules. Originally thought to be little more than reservoirs for cellular debris, the roles of exosomes regulating biological processes and in diseases are increasingly appreciated.
Several methods have been described for isolating exosomes from cellular culture media and biological fluids. Due to their small size and low density, differential ultracentrifugation and/or ultrafiltration are the most commonly used techniques for exosome isolation. However, plasma of HIV-1 infected individuals contains both exosomes and HIV viral particles, which are similar in size and density. Thus, efficient separation of exosomes from HIV viral particles in human plasma has been a challenge.
To address this limitation, we developed a procedure modified from Cantin et. al., 2008 for purification of exosomes from HIV particles in human plasma. Iodixanol velocity gradients were used to separate exosomes from HIV-1 particles in the plasma of HIV-1 positive individuals. Virus particles were identified by p24 ELISA. Exosomes were identified on the basis of exosome markers acetylcholinesterase (AChE), and the CD9, CD63, and CD45 antigens. Our gradient procedure yielded exosome preparations free of virus particles. The efficient purification of exosomes from human plasma enabled us to examine the content of plasma-derived exosomes and to investigate their immune modulatory potential and other biological functions.

Techniques describing a gradient procedure to separate exosomes from human immunodeficiency virus (HIV) particles are described. This procedure was used to isolate exosomes away from HIV particles in human plasma from HIV-infected individuals. The isolated exosomes were analyzed for cytokine/chemokine content.

HIV-1陽性個体の血漿からエキソソームの単離

Exosomes are small vesicles ranging in size from 30 nm to 100 nm that are released both constitutively and upon stimulation from a variety of cell types. ...

免疫・感染学

Isolation and Characterization of RNA-Containing Exosomes

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of endocytic origin were first proposed to function as a way for reticulocytes to eradicate the transferrin receptor while maturing into erythrocytes1, and were later named exosomes. Exosomes are formed by inward budding of late endosomes, producing multivesicular bodies (MVBs), and are released into the environment by fusion of the MVBs with the plasma membrane2. Since the first discovery of exosomes, a wide range of cells have been shown to release these vesicles. Exosomes have also been detected in several biological fluids, including plasma, nasal lavage fluid, saliva and breast milk3-6. Furthermore, it has been demonstrated that the content and function of exosomes depends on the originating cell and the conditions under which they are produced. A variety of functions have been demonstrated for exosomes, such as induction of tolerance against allergen7,8, eradication of established tumors in mice9, inhibition and activation of natural killer cells10-12, promotion of differentiation into T regulatory cells13, stimulation of T cell proliferation14 and induction of T cell apoptosis15. Year 2007 we demonstrated that exosomes released from mast cells contain messenger RNA (mRNA) and microRNA (miRNA), and that the RNA can be shuttled from one cell to another via exosomes. In the recipient cells, the mRNA shuttled by exosomes was shown to be translated into protein, suggesting a regulatory function of the transferred RNA16. Further, we have also shown that exosomes derived from cells grown under oxidative stress can induce tolerance against further stress in recipient cells and thus suggest a biological function of the exosomal shuttle RNA17. Cell culture media and biological fluids contain a mixture of vesicles and shed fragments. A high quality isolation method for exosomes, followed by characterization and identification of the exosomes and their content, is therefore crucial to distinguish exosomes from other vesicles and particles. Here, we present a method for the isolation of exosomes from both cell culture medium and body fluids. This isolation method is based on repeated centrifugation and filtration steps, followed by a final ultracentrifugation step in which the exosomes are pelleted. Important methods to identify the exosomes and characterize the exosomal morphology and protein content are highlighted, including electron microscopy, flow cytometry and Western blot. The purification of the total exosomal RNA is based on spin column chromatography and the exosomal RNA yield and size distribution is analyzed using a Bioanalyzer.

This paper demonstrates methods for the isolation, purification and detection of exosomes, as well as techniques for analysis of their molecular content. These methods are adaptable for exosome isolation from both cell culture media and biological fluids, and can beyond analysis of molecular content also be useful in functional studies.

RNA含有エキソソームの単離と解析

The field of exosome research is rapidly expanding, with a dramatic increase in publications in recent years. These small vesicles (30-100 nm) of ...

生物学

Isolation of Extracellular Vesicles from Murine Bronchoalveolar Lavage Fluid Using an Ultrafiltration Centrifugation Technique

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during physiological and pathological states. The isolation of EVs continues to be a major challenge in this field, due to limitations intrinsic to each technique. The differential ultracentrifugation with density gradient centrifugation method is a commonly used approach and is considered to be the gold standard procedure for EV isolation. However, this procedure is time-consuming, labor-intensive, and generally results in low scalability, which may not be suitable for small-volume samples such as bronchoalveolar lavage fluid. We demonstrate that an ultrafiltration centrifugation isolation method is simple and time- and labor-efficient yet provides a high recovery yield and purity. We propose that this isolation method could be an alternative approach that is suitable for EV isolation, particularly for small-volume biological specimens.

Here, we describe two extracellular vesicle isolation protocols, ultrafiltration centrifugation and ultracentrifugation with density gradient centrifugation, to isolate extracellular vesicles from murine bronchoalveolar lavage fluid samples. The extracellular vesicles derived from murine bronchoalveolar lavage fluid by both methods are quantified and characterized.

限外ろ過遠心分離法を用いたマウスの気管支肺胞洗浄液細胞小胞の分離

Extracellular vesicles (EVs) are newly discovered subcellular components that play important roles in many biological signaling functions during ...

Purification and microRNA Profiling of Exosomes Derived from Blood and Culture Media

Stable miRNAs are present in all body fluids and some circulating miRNAs are protected from degradation by sequestration in small vesicles called exosomes. Exosomes can fuse with the plasma membrane resulting in the transfer of RNA and proteins to the target cell. Their biological functions include immune response, antigen presentation, and intracellular communication. Delivery of miRNAs that can regulate gene expression in the recipient cells via blood has opened novel avenues for target intervention. In addition to offering a strategy for delivery of drugs or RNA therapeutic agents, exosomal contents can serve as biomarkers that can aid in diagnosis, determining treatment options and prognosis.	Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.	
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media.	
 Here we will describe the procedure for quantitatively analyzing miRNAs and messenger RNAs (mRNA) from exosomes secreted in blood and cell culture media. Purified exosomes will be characterized using western blot analysis for exosomal markers and PCR for mRNAs of interest. Transmission electron microscopy (TEM) and immunogold labeling will be used to validate exosomal morphology and integrity. Total RNA will be purified from these exosomes to ensure that we can study both mRNA and miRNA from the same sample. After validating RNA integrity by Bioanalyzer, we will perform a medium throughput quantitative real time PCR (qPCR) to identify the exosomal miRNA using Taqman Low Density Array (TLDA) cards and gene expression studies for transcripts of interest.
 These protocols can be used to quantify changes in exosomal miRNAs in patients, rodent models and cell culture media before and after pharmacological intervention. Exosomal contents vary due to the source of origin and the physiological conditions of cells that secrete exosomes. These variations can provide insight on how cells and systems cope with stress or physiological perturbations. Our representative data show variations in miRNAs present in exosomes purified from mouse blood, human blood and human cell culture media

The presence of stable microRNAs (miRNAs) in exosomes has generated immense interest as a novel mode of intercellular communication, for their potential utility as biomarkers and as a route for therapeutic intervention. Here we demonstrate exosome purification from blood and culture media followed by quantitative PCR to identify miRNAs being transported.

血と文化メディア由来エキソソームの精製およびマイクロRNAプロファイリング

Stable miRNAs are present in all body fluids and some circulating miRNAs  are protected from degradation by sequestration in small vesicles called  ...

Isolation of Small Noncoding RNAs from Human Serum

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are found in mammalian cells. These small RNAs are potent gene regulators controlling vital pathways such as growth, development and death and much interest has been directed at their expression in bodily fluids. This is due to their dysregulation in human diseases such as cancer and their potential application as serum biomarkers. However, the analysis of miRNA expression in serum may be problematic. In most cases the amount of serum is limiting and serum contains low amounts of total RNA, of which small RNAs only constitute 0.4-0.5%1. Thus the isolation of sufficient amounts of quality RNA from serum is a major challenge to researchers today. In this technical paper, we demonstrate a method which uses only 400 &#181;l of human serum to obtain sufficient RNA for either DNA arrays or qPCR analysis. The advantages of this method are its simplicity and ability to yield high quality RNA. It requires no specialized columns for purification of small RNAs and utilizes general reagents and hardware found in common laboratories. Our method utilizes a Phase Lock Gel to eliminate phenol contamination while at the same time yielding high quality RNA. We also introduce an additional step to further remove all contaminants during the isolation step. This protocol is very effective in isolating yields of total RNA of up to 100 ng/&#181;l from serum but can also be adapted for other biological tissues.

This protocol describes a method for extracting small RNAs from human serum. We have used this method to isolate microRNAs from cancer serum for use in DNA arrays and also singleplex quantitative PCR. The protocol utilizes phenol and guanidinium thiocyanate reagents with modifications to yield high quality RNA.

ヒト血清から小非コードRNAの分離

The analysis of RNA and its expression is a common feature in many laboratories. Of significance is the emergence of small RNAs like microRNAs, which are ...

Fast and Simplified Method for High Through-put Isolation of miRNA from Highly Purified High Density Lipoprotein

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for diagnosis and prognosis or may serve as targets for drug development, respectively. Recently it has been shown that miRNAs are carried in lipoproteins, particularly high density lipoproteins (HDL) and are delivered to recipient cells for uptake. This raises the possibility that miRNAs play a critical and pivotal role in cellular and organ function via regulation of gene expression as well as messenger for cell-cell communications and crosstalk between organs. Current methods for miRNA isolation from purified HDL are impractical when utilizing small samples on a large scale. This is largely due to the time consuming and laborious methods used for lipoprotein isolation. We have developed a simplified approach to rapidly isolate purified HDL suitable for miRNA analysis from plasma samples. This method should facilitate investigations into the role of miRNAs in health and disease and in particular provide new insights into the variety of biological functions, outside of the reverse cholesterol transport, that have been ascribed to HDL. Also, the miRNA species which are present in HDL can provide valuable information of clinical biomarkers for diagnosis of various diseases.

MicroRNAs play an important regulatory role and are emerging as novel therapeutic targets for various human diseases. It has been shown that miRNAs are carried in high density lipoproteins. We have developed a simplified method to rapidly isolate purified HDL suitable for miRNA analysis from human plasma.

高度に精製された高密度リポタンパク質からのmiRNAのハイスループット分離のための高速かつ簡便法

Small non-coding RNAs (miRNAs) have been implicated in a variety of human diseases including metabolic syndromes. They may be utilized as biomarkers for ...