我々は彼らの有益なコ​​メント、提案やそのプロトコルの共有のため建華玲、レイモンド·マクドナルド、ガルビンスウィフト、ミシェル·グリフィン、ポールGrippoとSatyanarayana Rachaganiに感謝します。この作品は、付与U01CA111294、オハイオ州立大学学内研究プログラムからのアイデア開発賞によってサポートされていました。

1。準備次のプラクティスは機関の動物実験委員会（IACUC）によって設定されたポリシーに準拠しています。 <ol><li>これは、任意の日にはもう6よりもマウスの膵臓を分離することをお勧めします。清潔でオートクレーブし、すべての手術器具、動物あたり1セットがあります。 </li><li>すべてのマイクロ遠心チューブにラベルを付けます。動物あたり4つの2 mlチューブ。 </li><li>氷上の50ml遠心管中で溶解試薬の8ミリリットルを配置します。注：精製キットは、膵臓の分離に適していますグアニジンチオシアネートフェノール試薬が付属していますが、溶解試薬があるため、分離ごとに必要な大量の使用されています。 </li><li>ホモジナイザーのブレードを洗浄するための15ml遠心チューブに以下の溶液約10mlを分注する。エタノール、70％（2本）、HPLCグレードの水（1管）とアウェイのRNaseの約200μLを含むHPLCグレードの水または同等のリボヌクレアーゼ不活性化。 </li></oL&gt; 2。手術、膵臓の解剖及び均質化<ol><li>コットンガーゼやペーパータオルを含むJARにイソフルラン1〜2mlのを配置します。マウスはイソフルランのソースに到達するのを防止するプラットフォーム上に配置されている。前各マウスを麻酔にジャーに新鮮イソフルランを追加します。 </li><li>優しくイソフルランを含むJARにマウスを置きます。 jarファイルに蓋をし、ゆっくりと前後​​に瓶を回転させることによって、麻酔の効果をモニターする。それはそれ自身で立つことができないになると動物に麻酔をかけている。 </li><li>麻酔の確認のために、ジャーから動物を削除して、しっかりと足指のピンチを適用します。マウスはつま先のピンチに反応しない場合は、2.5に進みます。 </li><li>マウスはつま先のピンチに反応する場合は、ステップ2.3に2.2を繰り返します。 </li><li>腹臥位で作業台の上に、麻酔した動物を配置します。しっかりと、マウスの子宮頸部に左手を置くことで、マウスの子宮頸部を脱臼。私達右手はマウスの尾をつかみ、素早く子宮頸部を脱臼に対して約上向きに45°の角度を引くING。 </li><li> 25ゲージの皮下注射針を使用して、平らな面に動物の四肢のそれぞれを貫通して表面（ペーパータオルで覆われた発泡スチロールのレベルピース）に死体を固定します。 </li><li>無菌手術器具を使用して、開いてすぐにマウスから膵臓を削除マウスの中央部をカット。膵臓を保持し、春のはさみを使用して、脾臓、小腸からそれをカットする鉗子を使用してください。マウスからの除去の間に膵臓を伸ばすないように注意してください。組織の破壊は、リボヌクレアーゼをリリースできた。注：マウスの​​膵臓を削除するのにかかる時間が非常に重要です。当業者は、約1分以下で膵臓を解剖することができるはずです。 </li><li>氷冷溶解試薬の8ミリリットルを含む50ミリリットルチューブに膵臓全体を置きます。チューブに蓋をし、簡単にTIを浸漬振る試薬にssue遅滞なく次のステップに進みます。組織への溶解試薬の比率が非常に重要です。溶解試薬の異なるボリュームを使用して単離されたRNAの完全性の比較の結果の項を参照してください。 </li><li> 5秒間均質化する。これは、組織が効率的な均質化のためにブレードに引き込まれることを可能にする遠心分離管の底部にホモジナイザーブレードを押圧することが重要である。 </li></ol>未消化の組織の3。取り外し注意：均質化に続いて、組織の小さなビットは溶解試薬に残ります。それはすぐに均質化し、RNAのリスク低下を介してではなく、未消化いくつかの組織を残して組織を溶解することがより重要である。 <ol><li>転送2ミリリットルの微量遠心チューブに溶解した膵臓を含む溶解試薬の2ミリリットル。アイソレーションごとホモジネートの2マイクロチューブを収集します。今後の研究のために-80℃で、残りのホモジネートまたはストアを破棄します。 </li><li>セント4℃で、未消化の組織をペレット化する5分間12,000×gでrifuge。これはおそらく、リボヌクレアーゼ（ステップ5.13）で試料を汚染するRNAのその後のソリューションに未消化の組織のキャリーオーバーな​​どの重要なステップです。 </li><li> 2ミリリットルのマイクロ遠心チューブに移す上清の1ミリリットル。すぐに分離を進め、将来の使用のために-80℃で複製チューブを格納します。次のステップに進むながら濡れた氷の上にホモジネートを含むすべてのチューブを配置します。 </li><li>適切な化学廃棄物容器に残った溶解試薬を処分。 </li></ol>ホモジナイザーブレードの4。クリーニング<ol><li>次の単離に進む前に、70％エタノール10ml中に置くことにより、ホモジナイザーをクリーニングブレード。刃に巻き込まれることがあり、組織の任意の塊を除去するために20秒程度のためのホモジナイザーをアクティブにします。 </li><li>ホモジナイザーブレードに残っている部分を除去するために200μlのピペットチップを使用してください。 </lI&gt; <li>水、一般的なRNA分解酵素を不活性化し、70％エタノールでの手順を繰り返し4.1。キムワイプクリーンでホモジナイザーシャフトを乾燥させます。 </li><li>残りの動物の膵臓の分離について、手順2.1、むしろRNAをプールとは別の各動物から単離されたRNAを保つ。 </li></ol> 5。RNAの単離次のセクションでは、精製キットプロトコルと同じです。精製キットの利点は、低分子RNAを含む全RNAを単離することである。 <ol><li>凍結したホモジネート溶液を使用した場合、5分間RTでスタンドを濡れた氷の上で解凍してみましょう。 </li><li> 200μlのクロロホルムを加え、チューブにキャップをし、15秒間、手でよく振る。チューブは2〜3分間室温で放置。 </li><li> 12,000 XG、4℃で15分間遠心</li><li>マイクロ遠心チューブに上層の水層を転送します。イソプロパノール1.5ボリュームを追加し、上下に数回ピペッティングしてよく混ぜ。 </li><li> 70まで転送する2 mlのコレクションチューブに配置されているスピンカラムへの試料液の0。室温で15秒間8000×gで蓋をし、遠心分離機を閉じます。 </li><li>フロースルーを捨てる。 </li><li>繰り返してサンプルの残りを使って、5.5から5.6を繰り返します。 </li><li>スピンカラムに700μlのBuffer RWTを追加します。 8,000 X gで15秒間遠心します。フロースルーを捨てる。 </li><li>スピンカラムにピペットで500μlのBuffer RPE。カラムを洗浄するために8000×gで15秒間遠心分離する。フロースルーを捨てる。 </li><li>スピンカラムに500μlのBuffer RPEを追加します。カラムを乾燥するために8000×gで2分間遠心分離する。 </li><li> 1.5 mlのマイクロ遠心チューブにスピンカラムを移す。直接スピンカラム·メンブレンにピペットで80μlのリボヌクレアーゼを含まない水。 RNAを溶出するために8000×gで1分間遠心分離する。 </li><li> RNA溶液に1μlのRNase阻害剤を追加します。 -80℃で保存するか、5.13に進みます。 </li><li> RNAの完全性を検証するために、最初のRNA conceを計算分光光度計を用いてntrationと吸光度比260/280。 </li><li>分子生物学グレードの水で約500 ngの/μlにRNA溶液の一部を希釈する。 </li><li>キャピラリー電気泳動分析を用いてRNA完全性（ すなわち 、RIN）を決定します。 RNA溶液約5μlを分析のために十分である。 </li><li> -80℃でRNA溶液（ 図2を参照）、店舗を分取</li></ol>

<ol>
	<li>Chen, C., Ridzon, D. A., Broomer, A. J., Zhou, Z., Lee, D. H., Nguyen, J. T., Barbisin, M., Xu, N. L., Mahuvakar, V. R., Andersen, M. R., 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=Real-time+quantification+of+microRNAs+by+stem-loop+RT-PCR.">Real-time quantification of microRNAs by stem-loop RT-PCR.</a> Nucleic Acids Res. 33, e179 (2005).</li><li>Chirgwin, J. M., Przybyla, A. E., MacDonald, R. J., Rutter, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+biologically+active+ribonucleic+acid+from+sources+enriched+in+ribonuclease.">Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease.</a> Biochemistry. 18, 5294-5299 (1979).</li><li>Chomczynski, P., Sacchi, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-step+method+of+RNA+isolation+by+acid+guanidinium+thiocyanate-phenol-chloroform+extraction.">Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.</a> Anal Biochem. 162, 156-159 (1987).</li><li>Griffin, M., Abu-El-Haija, M., Abu-El-Haija, M., Rokhlina, T., Uc, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simplified+and+versatile+method+for+isolation+of+high-quality+RNA+from+pancreas.">Simplified and versatile method for isolation of high-quality RNA from pancreas.</a> Biotechniques. 52, 332-334 (2012).</li><li>Iyer, V. R., Eisen, M. B., Ross, D. T., Schuler, G., Moore, T., Lee, J. C., Trent, J. M., Staudt, L. M., Hudson Jr, J., Boguski, M. S., 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=The+transcriptional+program+in+the+response+of+human+fibroblasts+to+serum.">The transcriptional program in the response of human fibroblasts to serum.</a> Science. 283, 83-87 (1999).</li><li>Lenstra, J. A., Beintema, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+amino+acid+sequence+of+mouse+pancreatic+ribonuclease.+Extremely+rapid+evolutionary+rates+of+the+myomorph+rodent+ribonucleases.">The amino acid sequence of mouse pancreatic ribonuclease. Extremely rapid evolutionary rates of the myomorph rodent ribonucleases.</a> European journal of biochemistry FEBS. 98, 399-408 (1979).</li><li>Mullin, A. E., Soukatcheva, G., Verchere, C. B., Chantler, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+in+situ+ductal+perfusion+to+facilitate+isolation+of+high-quality+RNA+from+mouse+pancreas.">Application of in situ ductal perfusion to facilitate isolation of high-quality RNA from mouse pancreas.</a> Biotechniques. 40, 617-621 (2006).</li><li>Rio, D. C., Ares, M., Hannon, G. J., Nilsen, T. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> RNA A Laboratory Manual. , (2011).</li><li>Streit, S., Michalski, C. W., Erkan, M., Kleeff, J., Friess, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Northern+blot+analysis+for+detection+and+quantification+of+RNA+in+pancreatic+cancer+cells+and+tissues.">Northern blot analysis for detection and quantification of RNA in pancreatic cancer cells and tissues.</a> Nat Protoc. 4, 37-43 (2009).</li><li>Zmuda, E. J., Powell, C. A., Hai, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+method+for+murine+islet+isolation+and+subcapsular+kidney+transplantation.">A method for murine islet isolation and subcapsular kidney transplantation.</a> Journal of Visualized Experiments JoVE. , (2011).</li></ol>

The authors have nothing to disclose.

リボヌクレアーゼが豊富な組織からRNAを単離することは分子生物学的実験のための大きな課題を表しています。様々な試薬およびキットは、主にRNA抽出のフェノールグアニジンチオシアネート法に基づいているが市販されている。グアニジンチオシアネートは、タンパク質を変性し、このようにリボヌクレアーゼ活性を低下させる。しかし、膵臓リボヌクレアーゼの膨大な大きさは、RNA単離?...

高い整合性を持つRNAの単離は、ノーザンブロット法9、定量RT-PCRの1または5の遺伝子発現プロファイリングなどの日常的な分子生物学の実験のために必要とされている。 RNA単離の最も現代的な方法は、グアニジンチオシアネートプロトコル2、3の修正に基づいている。グアニジンチオシアネートは、強力なタンパク質変性剤であり、効果的に、最も条件の下でリボヌクレアーゼの活性を破壊します。をChomczynskiおよびSacchi 3の一般的な方法は、約4時間までの単離時間を短縮する、チオシアン酸グアニジン溶解溶液にフェノールを組み合わせる。多くの市販のRNA抽出試薬をChomczynskiおよびSacchi法3に基づいている。 例えば、ヒトやマウスの膵臓リボヌクレアーゼ多い組織は、RNAを分離するための新たな課題が発生します。マウスの膵臓は、そのうちのいくつかは、ドゥリンリリースされるリボヌクレアーゼ6の75ミリグラムを含有することができるg組織の自己消化、その結果膵臓組織の破壊。グアニジンチオシアプロトコルの改変に成功し、高い完全性2、4、7、10の膵臓からRNAを単離するために使用されてきた。RNA安定化試薬の注入マウスの膵臓中へ、高い整合性4、7、10の一方注入をRNAの単離を容易に膵臓へのソリューションは素晴らしいスキルを必要とし、このような解剖顕微鏡などの特殊な機器を必要とし、処置の間に組織構造を崩壊させることは、細胞溶解の原因となります。 RNA安定化試薬を用いて組織を灌流すると、タンパク質の単離と組織染色など、他のアプリケーションに干渉することがあります。さらに、この技術は、ヒト膵臓からRNAを単離するには適していない。他のプロトコルは、研究者2による特定の溶液の調製を必要とする。 我々は、マウスの膵臓からの高い整合性RNAを単離するためのプロトコルを報告する。目Eプロトコルは、以前に発表された方法の要素を使用しており、大部分は、標準的なフェノール/グアニジンチオシアネート·ベースの溶解試薬プロトコルに基づいています。これは、専門の溶液の調製を必要とせず、また膵臓に試薬の注入を必要としません。成功したRNAの単離のための重要なステップは、未消化組織の除去は、得られたRNA溶液にリボヌクレアーゼ阻害剤の分離および包含を位相の前に、組織比に高いフェノール/グアニジン系、溶解試薬を含む。これらおよび他の変更を使用して、我々は、典型的には、7より大きいルーチン遺伝子発現解析に用いるRINでRNAを単離する。

<table border="0" cellpadding="0" cellspacing="0" width="788">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Name of Reagent/ Equipment</td>
			<td style="width:93px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:360px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Power Gen 500&#160;</td>
			<td style="width:93px;">Fisher Scientific</td>
			<td style="width:119px;">14-261-03&#160;</td>
			<td>Homogenizer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">5424R</td>
			<td style="width:93px;">Eppendorf</td>
			<td style="width:119px;"></td>
			<td>Refrigerated microcentifuge</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trizol reagent</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;">15596018</td>
			<td>Lysis reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Student Vannas spring scissors</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">91500-09&#160;</td>
			<td>use to cut pancreas from attached tissues</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Surgical scissors, 6 inch</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">08-951-20</td>
			<td>use to cut scin of mouse</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blunt end forceps</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">1381239</td>
			<td>use to hold pancreas while dissecting</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Isoflurane USP</td>
			<td style="width:93px;">Abbott Labs</td>
			<td style="width:119px;">4/8/5210</td>
			<td>Anesthetic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Rnase out (40 U/&#181;l)</td>
			<td style="width:93px;">Invitrogen</td>
			<td style="width:119px;">10777-019</td>
			<td>Rnase inhbitor</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;width:216px;">Rnase Away</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;">10328-011</td>
			<td>General Rnase inactivator</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">HPLC grade water</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">W5-4</td>
			<td>For washing homogenizer blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Molecular Biology grade water</td>
			<td style="width:93px;">Hyclone</td>
			<td style="width:119px;">SH30538.03</td>
			<td>Elution of RNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">miRNeasy Mini kit</td>
			<td style="width:93px;">Qiagen</td>
			<td style="width:119px;">217004</td>
			<td>Purification Kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">2 ml microcentrifuge tubes, certified Rnase Dnase free</td>
			<td style="width:93px;">USA Scientific Plastics</td>
			<td style="width:119px;">1620-2700</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">15 ml centrifuge tubes</td>
			<td style="width:93px;">Falcon</td>
			<td style="width:119px;">352099</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">70% ethanol</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">100% ethanol</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">200 ul pipet tips</td>
			<td style="width:93px;">Rainin</td>
			<td style="width:119px;">GPL200F</td>
			<td>For cleaning homogenizer blades</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform</td>
			<td style="width:93px;">Fisher</td>
			<td style="width:119px;">BP1145-1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nanodrop</td>
			<td style="width:93px;">Thermo</td>
			<td style="width:119px;">ND-1000</td>
			<td>Spectrophotometer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bioanalyzer</td>
			<td style="width:93px;">Agilent</td>
			<td style="width:119px;"></td>
			<td>Capillary electrophoresis analyzer to measue RNA integrity</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNAlater</td>
			<td style="width:93px;">Ambion</td>
			<td style="width:119px;"></td>
			<td>RNA Stabilization Reagent&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNeasy&#160;spin columns&#160;</td>
			<td style="width:93px;">Qiagen</td>
			<td style="width:119px;"></td>
			<td>&#160;spin columns&#160;as a part of the miRNeasy Mini kit</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mice</td>
			<td style="width:93px;">Chales River</td>
			<td style="width:119px;">strain C57BL/6</td>
			<td style="width:360px;">Their age ranged from 4 to 12 months.&#160;&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"></td>
			<td style="width:93px;"></td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Mice&#160;</td>
			<td style="width:93px;">Jackson Lab</td>
			<td style="width:119px;">strain 129</td>
			<td style="width:360px;">Their age ranged from 4 to 12 months.&#160;&#160;</td>
		</tr>
	</tbody>
</table>

rna isolation from mouse pancreas: a ribonuclease-rich tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

溶解ホモジネートの1ミリリットルからの全RNAの収量は20〜40μgのです。 OD 280分の260の比率は、典型的には約2.0であり、RINは7.0よりも一貫して大きい。 RINは6≤されている場合は、アイソレーションが繰り返される必要があります。時々、8.0より高いRINが達成される。 前膵臓の除去にマウスを安楽死させる2つの最も一般的に使用されている方法は、CO 2窒息または?...

Watch this Scientific Journal Video about RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue at JoVE.com

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

マウス膵臓からのRNAの単離：リボヌクレアーゼが豊富な組織

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

rna-isolation-from-mouse-pancreas-a-ribonuclease-rich-tissue

Research

JoVE Journal

Biology

27.4K ビュー.  The Ohio State University. We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

ビデオ: RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Neuronal Nuclei Isolation from Human Postmortem Brain Tissue

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, little is known about changes in neuronal chromatin and nuclear organization during the course of development and aging, or in chronic neuropsychiatric disease. However, to date most chromatin and DNA based assays (other than FISH) lack single cell resolution. To this end, the considerable cellular heterogeneity of brain tissue poses a significant limitation, because typically various subpopulations of neurons are intermingled with different types of glia and other non-neuronal cells. One possible solution would be to grow cell-type specific cultures, but most CNS cells, including neurons, are ex vivo sustainable, at best, for only a few weeks and thus would provide an incomplete model for epigenetic mechanisms potentially operating across the full lifespan.  Here, we provide a protocol to extract and purify nuclei from frozen (never fixed) human postmortem brain. The method involves extraction of nuclei in hypotonic lysis buffer, followed by ultracentrifugation and immunotagging with anti-NeuN antibody. Labeled neuronal nuclei are then collected separately using fluorescence-activated sorting. This method should be applicable to any brain region in a wide range of species and suitable for chromatin immunoprecipitation studies with site- and modification-specific anti-histone antibodies, and for DNA methylation and other assays. 

The cellular heterogeneity of brain tissue poses a significant limitation for the study of epigenetic markings in chromatin because most assays lack single cell resolution. Neurons typically are intermingled with glia and other non-neuronal cells. We provide a protocol to extract and collect neuronal nuclei from human brain.

人間の死後の脳組織から神経細胞核の単離

Neurons in the human brain become postmitotic largely during prenatal development, and thus maintain their nuclei throughout the full lifespan. However, ...

生物学

Isolation of Primary Myofibroblasts from Mouse and Human Colon Tissue

The myofibroblast is a stromal cell of the gastrointestinal (GI) tract that has been gaining considerable attention for its critical role in many GI functions. While several myofibroblast cell lines are commercially available to study these cells in vitro, research results from a cell line exposed to experimental cell culture conditions have inherent limitations due to the overly reductionist nature of the work. Use of primary myofibroblasts offers a great advantage in terms of confirming experimental findings identified in a cell line. Isolation of primary myofibroblasts from an animal model allows for the study of myofibroblasts under conditions that more closely mimic the disease state being studied. Isolation of primary myofibroblasts from human colon tissue provides arguably the most relevant experimental data, since the cells come directly from patients with the underlying disease. We describe a well-established technique that can be utilized to isolate primary myofibroblasts from both mouse and human colon tissue. These isolated cells have been characterized to be alpha-smooth muscle actin and vimentin-positive, and desmin-negative, consistent with subepithelial intestinal myofibroblasts. Primary myofibroblast cells can be grown in cell culture and used for experimental purposes over a limited number of passages.

The myofibroblast is an influential stromal cell of the gastrointestinal tract that regulates important physiologic processes in both normal and disease states. We describe a technique that allows for the isolation of primary myofibroblasts from both mouse and human colon tissue, which can be utilized for in vitro experimentation.

マウスおよびヒト結腸組織からの一次筋線維芽細胞の分離

The myofibroblast is  a stromal cell of the gastrointestinal (GI) tract that has been gaining  considerable attention for its critical role in many GI ...

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the development of protocols to produce pancreatic cells from stem cells 1. The whole embryonic organ can be cultured at multiple stages of development 2-4. These culture methods have been useful to test drugs and to image developmental processes. However the expansion of the organ is very limited and morphogenesis is not faithfully recapitulated since the organ flattens. 
We propose three-dimensional (3D) culture conditions that enable the efficient expansion of dissociated mouse embryonic pancreatic progenitors. By manipulating the composition of the culture medium it is possible to generate either hollow spheres, mainly composed of pancreatic progenitors expanding in their initial state, or, complex organoids which progress to more mature expanding progenitors and differentiate into endocrine, acinar and ductal cells and which spontaneously self-organize to resemble the embryonic pancreas. 
We show here that the in vitro process recapitulates many aspects of natural pancreas development. This culture system is suitable to investigate how cells cooperate to form an organ by reducing its initial complexity to few progenitors. It is a model that reproduces the 3D architecture of the pancreas and that is therefore useful to study morphogenesis, including polarization of epithelial structures and branching. It is also appropriate to assess the response to mechanical cues of the niche such as stiffness and the effects on cell&#180;s tensegrity.

In Vitro Pancreas Organogenesis from Dispersed Mouse Embryonic Progenitors

The three-dimensional culture method described in this protocol recapitulates pancreas development from dispersed embryonic mouse pancreas progenitors, including their substantial expansion, differentiation and morphogenesis into a branched organ. This method is amenable to imaging, functional interference and manipulation of the niche.

分散マウス胚性前駆細胞からin vitro膵臓器官形成

The pancreas is an essential organ that regulates glucose homeostasis and secretes digestive enzymes. Research on pancreas embryogenesis has led to the ...

Protein Isolation from the Developing Embryonic Mouse Heart Valve Region

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis method may not be sufficiently sensitive to detect a protein of interest. To overcome these difficulties, we examined protocols for obtaining protein from adult human cardiac valves and modified these protocols for the developing early embryonic mouse counterparts. In brief, the mouse embryonic aortic valve regions, including the aortic valve and surrounding aortic wall, are collected in the minimal possible volume of a Tris-based lysis buffer with protease inhibitors. If required based on the breeding strategy, embryos are genotyped prior to pooling four embryonic aortic valve regions for homogenization. After homogenization, an SDS-based sample buffer is used to denature the sample for running on an SDS-PAGE gel and subsequent western blot analysis. Although the protein concentration remains too low to quantify using spectrophotometric protein quantification assays and have sample remaining for subsequent analyses, this technique can be used to successfully detect and semi-quantify phosphorylated proteins via western blot from pooled samples of four embryonic day 13.5 mouse aortic valve regions, each of which yields approximately 1 &#956;g of protein. This technique will be of benefit for studying cell signaling pathway activation and protein expression levels during early embryonic mouse valve development.

The analysis of protein expression in young embryonic mouse valves has been hampered by the limited tissue available. This manuscript provides a protocol for preparing protein from developing embryonic mouse valve regions for western blot analysis.

途上胚性マウス心臓弁地域からのタンパク質の単離

Western blot analysis is a commonly employed technique for detecting and quantifying protein levels. However, for small tissue samples, this analysis ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

培養細胞およびマウス組織から単離とミトコンドリアの機能解析

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Isolation and Culture of Primary Mouse Keratinocytes from Neonatal and Adult Mouse Skin

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin defense by forming an intact skin barrier against environmental insults, such as UVB irradiation or pathogens, and also by initiating an inflammatory response upon those insults. Here we describe methods to isolate KCs from neonatal mouse skin and from adult mouse tail skin. We also describe culturing conditions using defined growth supplements (dGS) in comparison to chelexed fetal bovine serum (cFBS). Functionally, we show that both neonatal and adult KCs are highly responsive to high calcium-induced terminal differentiation, tight junction formation and stratification. Additionally, cultured adult KCs are susceptible to UVB-triggered cell death and can release large amounts of TNF upon UVB irradiation. Together, the methods described here will be useful to researchers for the setup of in vitro models to study epidermal biology in the neonatal mouse and/or the adult mouse.

Epidermal keratinocytes form a functional skin barrier and are positioned at the front line of host defense against external environmental insults. Here we describe methods for isolation and primary culture of epidermal keratinocytes from neonatal and adult mouse skin, and induction of terminal differentiation and UVB-triggered inflammatory response from keratinocytes.

新生児および成人マウス皮膚由来の初代マウスケラチノサイトの単離および培養

The keratinocyte (KC) is the predominant cell type in the epidermis, the outermost layer of the skin. Epidermal KCs play a critical role in providing skin ...

Nuclei Isolation from Whole Tissue using a Detergent and Enzyme-Free Method

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension with&#160;intact cell or nuclei involves dissociation and permeabilization, steps that can introduce unwanted noise and undesirable damage. Particularly, certain cell-types such as neurons are challenging to dissociate into individual cells. Additionally, permeabilization of the cellular membrane to release nuclei requires optimization by trial-and-error, which can be time consuming, labor intensive and financially nonviable. To enhance the robustness and reproducibility of sample preparation for high-throughput sequencing, we describe a rapid enzyme and detergent-free column-based nuclei isolation method. The protocol enables efficient isolation of nuclei from the entire zebrafish brain within 20 minutes. The isolated nuclei display intact nuclear morphology and low propensity to aggregate. Further, flow cytometry allows nuclei enrichment and clearance of cellular debris for downstream application. The protocol, which should work on soft tissues and cultured cells, provides a simple and accessible method for sample preparation that can be utilized for high-throughput profiling, simplifying the steps required for successful single-nuclei RNA-seq and ATAC-seq experiments.

Single nuclei isolation relies on dissociation and detergent-based permeabilization of the cell membrane, steps that need optimization and are prone to introducing technical artifacts. We demonstrate a detergent and enzyme-free protocol for rapid isolation of intact nuclei directly from whole tissue, yielding nuclei suitable for single-nucleus RNA-seq (snRNA-Seq) or ATAC-seq.

洗浄剤と酵素を含まない方法を用いた組織全体からの核分離

High-throughput transcriptome and epigenome profiling requires preparation of a single cell or single nuclei suspension. Preparation of the suspension ...

Tissue Collection and RNA Extraction from the Human Osteoarthritic Knee Joint

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty (TKA) is a common surgical intervention. Experiments using primary human OA tissues obtained from TKA provide the capability to investigate disease mechanisms ex vivo. While OA was previously thought to impact mainly the cartilage, it is now known to impact multiple tissues in the joint. This protocol describes patient selection, sample processing, tissue homogenization, RNA extraction, and quality control (based on RNA purity, integrity, and yield) from each of seven unique tissues to support disease mechanism investigation in the knee joint. With informed consent, samples were obtained from patients undergoing TKA for OA. Tissues were dissected, washed, and stored within 4 h of surgery by flash freezing for RNA or formalin fixation for histology. Collected tissues included articular cartilage, subchondral bone, meniscus, infrapatellar fat pad, anterior cruciate ligament, synovium, and vastus medialis oblique muscle. RNA extraction protocols were tested for each tissue type. The most significant modification involved the method of disintegration used for low-cell, high-matrix, hard tissues (considered as cartilage, bone, and meniscus) versus relatively high-cell, low-matrix, soft tissues (considered as fat pad, ligament, synovium, and muscle). It was found that pulverization was appropriate for hard tissues, and homogenization was appropriate for soft tissues. A proclivity for some subjects to yield higher RNA integrity number (RIN) values than other subjects consistently across multiple tissues was observed, suggesting that underlying factors such as disease severity may impact RNA quality. The ability to isolate high-quality RNA from primary human OA tissues provides a physiologically relevant model for sophisticated gene expression experiments, including sequencing, that can lead to clinical insights that are more readily translated to patients.

Primary tissues obtained from patients following total knee arthroplasty provide an experimental model for osteoarthritis research with maximal clinical translatability. This protocol describes how to identify, process, and isolate RNA from seven unique knee tissues to support mechanistic investigation in human osteoarthritis.

ヒト変形性膝関節からの組織採取とRNA抽出

Osteoarthritis (OA) is a chronic and degenerative joint disease most often affecting the knee. As there is currently no cure, total knee arthroplasty ...

Nuclei Isolation from Adult Mouse Kidney for Single-Nucleus RNA-Sequencing

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are executed by multiple cell types arranged in a complex architecture across the corticomedullary axis of the organ. Recent advances in single-cell transcriptomics have accelerated the understanding of cell type-specific gene expression in renal physiology and disease. However, enzyme-based tissue dissociation protocols, which are frequently utilized for single-cell RNA-sequencing (scRNA-seq), require mostly fresh (non-archived) tissue, introduce transcriptional stress responses, and favor the selection of abundant cell types of the kidney cortex resulting in an underrepresentation of cells of the medulla.
Here, we present a protocol that avoids these problems. The protocol is based on nuclei isolation at 4 &#176;C from frozen kidney tissue. Nuclei are isolated from a central piece of the mouse kidney comprised of the cortex, outer medulla, and inner medulla. This reduces the overrepresentation of cortical cells typical for whole-kidney samples for the benefit of medullary cells such that data will represent the entire corticomedullary axis at sufficient abundance. The protocol is simple, rapid, and adaptable and provides a step towards the standardization of single-nuclei transcriptomics in kidney research.

Here, we present a protocol to isolate high-quality nuclei from frozen mouse kidneys that improve the representation of medullary kidney cell types and avoids the gene expression artifacts from enzymatic tissue dissociation.

単核RNAシーケンシングのための成体マウス腎臓からの核分離

The kidneys regulate diverse biological processes such as water, electrolyte, and acid-base homeostasis. Physiological functions of the kidney are ...

Isolation, Characterization, and Therapeutic Application of Extracellular Vesicles from Cultured Human Mesenchymal Stem Cells

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological regulators of organismal homeostasis and important indicators of pathologies; in the meantime, their immense potential to establish accessible and controllable disease therapeutics is emerging. Mesenchymal stem cells (MSCs) can release large amounts of EVs in culture, which have shown promise to jumpstart effective tissue regeneration and facilitate extensive therapeutic applications with good scalability and reproducibility. There is a growing demand for simple and effective protocols for collecting and applying MSC-EVs. Here, a detailed protocol is provided based on differential centrifugation to isolate and characterize representative EVs from cultured human MSCs, exosomes, and microvesicles for further applications. The adaptability of this method is shown for a series of downstream approaches, such as labeling, local transplantation, and systemic injection. The implementation of this procedure will address the need for simple and reliable MSC-EVs collection and application in translational research.

The present protocol describes the differential centrifugation for isolating and characterizing representative EVs (exosomes and microvesicles) from cultured human MSCs. Further applications of these EVs are also explained in this article.

培養ヒト間葉系幹細胞からの細胞外小胞の単離、特性評価、および治療応用

Extracellular vesicles (EVs) are heterogeneous membrane nanoparticles released by most cell types, and they are increasingly recognized as physiological ...