私たちは、ゲノム機能施設と分光とバイオイメージング施設、基生研中核研究施設、およびモデル植物研究施設、基生研バイオリソースセンターのスタッフに感謝します。

植物の種子からの全RNAの1の抽出<ol><li>バッファセット、スピンカラム、1.5および2.0 mLのポリプロピレンチューブ、およびヌクレアーゼフリー1.5 mLのポリプロピレンチューブを準備します。 </li><li> RNA抽出、激しくボルテックスするための溶解緩衝液をセルに（以下、PVPという）分子生物学グレードのポリビニルピロリドン（w / v）の1％を加えます。完全に溶解するために25℃で20分間インキュベートします。 20分間のインキュベーション後、気泡の形成を防止するために逆さまにチューブを回転させることによって穏やかにバッファーを混合します。使用前に室温（15~25℃）で保管してください。 </li><li>氷上で1.5 mLのポリプロピレンチューブ中のシロイヌナズナと場所から収穫果物。アルミプレート上に果物を置き、4℃で維持し、実体顕微鏡下で果実から種子を分離します。 <ol><li>氷の上にアルミラックに格納され、直ちに液体窒素中にチューブを配置された1.5 mLのポリプロピレンチューブに分離された種子（約200種を）置きますitrogen。 </li></ol></li><li>液体窒素からチューブを外して、氷の上にアルミニウムラックに戻します。 4℃で千×gで1分間、1％のPVPと遠心分離機を含む緩衝液100μLを加えます。 </li><li>氷の上にアルミラックにチューブを保持したまま60秒間モーターグラインダーを使用して、ステンレス製の乳棒でサンプルをホモジナイズします。 </li><li> 1％のPVPを含有する緩衝液550μLを加える逆さまチューブを回して穏やかにバッファーを混合し、25℃で10分間インキュベートします。 </li><li> 25℃で8000×gで5分間遠心し、新しい1.5 mLのポリプロピレンチューブに上清550μLを移します。 </li><li> 25°C 10,000×gで5分間遠心し、新しい1.5 mLのポリプロピレンチューブに上清450μLを移します。 </li><li> RNA抽出のための細胞溶解液として上清を使用してください。以下では、市販のキットには、メーカーの説明書に記載の手順に従ってください。 </li><li>溶出低レベルの転写物の発現パターンをモニターするためのRNAの十分に高い濃度を確実にするために、溶出緩衝液の最小量を使用してRNA。使用するまでディープフリーザーでRNAを保管してください。 </li></ol> RNAの品質の2.確認<ol><li> 、全RNAを解凍穏やかなタッピングによって混合し、氷上でアルミラックにチューブを配置します。 </li><li>微量分光光度計を用いてRNA濃度、A260 / A280比、およびA260 / A230比を測定します。 </li><li> 70 ngの/μLの最終濃度になるようにRNaseフリー水で全RNAを希釈します。 </li></ol>種子からの全RNAの3逆転写<ol><li>市販のキットから全RNAおよびバッファセットを解凍します。穏やかなタッピングした後、氷上でキットからの酵素を保管してください。ヌクレアーゼフリー0.2 mlのポリプロピレンチューブを準備します。 </li><li> 0.2 mLのpolypropyに全RNAの5μL、オリゴdTプライマーの1μL（50μM）、およびランダム6量体の1μM（50μM）を追加氷上のレンチューブ。 </li><li> 37ºCで15分間インキュベートし、氷上に置きます。 </li><li>以下は、逆転写PCRキットで、製造業者の説明書に記載されている手順に従ってください。 </li><li>使用前に氷上で逆転写産物を置きます。 </li></ol> 4.定量的リアルタイムPCR解析<ol><li>製造業者のプロトコルを使用して、標的遺伝子配列を保有するプラスミドを構築します。 </li><li>標準曲線のためのDNAテンプレートの100-500,000コピー/μLに濃度を調整します。 </li><li>蒸留水で：（100 1）のcDNAのソリューションを希釈します。 </li><li>定量的リアルタイムPCRキットからマスターミックスに希釈したcDNA溶液及び標準曲線のためのプラスミドの2μLを追加します。 </li><li> 35秒30秒95℃をした後、5秒、95℃で40サイクル、60℃：以下のサイクリング条件を用いてリアルタイムPCRを設定します。 </li><li>男に従ってコピー数を分析リアルタイムPCRシステム用のufacturerの指示。 </li></ol>

<ol>
	<li>Hills, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+storage-product+synthesis+in+seeds.">Control of storage-product synthesis in seeds.</a> Curr Opin Plant Biol. 7 (3), 302-308 (2004).</li><li>Li-Beisson, Y., 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=Acyl-lipid+metabolism.">Acyl-lipid metabolism.</a> Arabidopsis Book. 11, e0161 (2013).</li><li>Bates, P. D., Stymne, S., Ohlrogge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+pathways+in+seed+oil+synthesis.">Biochemical pathways in seed oil synthesis.</a> Curr Opin Plant Biol. 16 (3), 358-364 (2013).</li><li>Santos-Mendoza, M., 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=Deciphering+gene+regulatory+networks+that+control+seed+development+and+maturation+in+Arabidopsis.">Deciphering gene regulatory networks that control seed development and maturation in Arabidopsis.</a> Plant J. 54 (4), 608-620 (2008).</li><li>Durrett, T. P., Benning, C., Ohlrogge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+triacylglycerols+as+feedstocks+for+the+production+of+biofuels.">Plant triacylglycerols as feedstocks for the production of biofuels.</a> Plant J. 54 (4), 593-607 (2008).</li><li>Kanai, M., 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+Plastidic+DEAD-box+RNA+helicase+22,+HS3,+is+essential+for+plastid+functions+both+in+seed+development+and+in+seedling+growth.">The Plastidic DEAD-box RNA helicase 22, HS3, is essential for plastid functions both in seed development and in seedling growth.</a> Plant Cell Physiol. 54 (9), 1431-1440 (2013).</li><li>Kanai, M., 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=Extension+of+oil+biosynthesis+during+the+mid-phase+of+seed+development+enhances+oil+content+in+Arabidopsis+seeds.">Extension of oil biosynthesis during the mid-phase of seed development enhances oil content in Arabidopsis seeds.</a> Plant Biotechnol J. 14 (5), 1241-1250 (2016).</li><li>Dekkers, B. J., 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=Identification+of+reference+genes+for+RT-qPCR+expression+analysis+in+Arabidopsis+and+tomato+seeds.">Identification of reference genes for RT-qPCR expression analysis in Arabidopsis and tomato seeds.</a> Plant Cell Physiol. 53 (1), 28-37 (2012).</li><li>Salzman, R. A., 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=An+improved+RNA+isolation+method+for+plant+tissues+containing+high+levels+of+phenolic+compounds+or+carbohydrates.">An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates.</a> Plant Mol Biol Rep. 17 (1), 11-17 (1999).</li><li>Vicient, C. M., Delseny, M. <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+total+RNA+from+Arabidopsis+thaliana+seeds.">Isolation of total RNA from Arabidopsis thaliana seeds.</a> Anal Biochem. 268 (2), 412-413 (1999).</li><li>Wang, G. F., 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=Isolation+of+high+quality+RNA+from+cereal+seeds+containing+high+levels+of+starch.">Isolation of high quality RNA from cereal seeds containing high levels of starch.</a> Phytochem Analysis. 23 (2), 159-163 (2012).</li><li>Birtic, S., Kranner, I. <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+high-quality+RNA+from+polyphenol-,+polysaccharide-+and+lipid-rich+seeds.">Isolation of high-quality RNA from polyphenol-, polysaccharide- and lipid-rich seeds.</a> Phytochem Analysis. 17 (3), 144-148 (2006).</li></ol>

The authors have nothing to disclose.

遺伝子発現プロファイルは、植物生理学の我々の理解に貢献します。従って、特定のRNAの単離方法は、各サンプル条件9-12のために開発されています。我々は、種子からのRNA単離の間に抑制されたプロセスを調査し、RNAをシリカ膜への結合が厳しく抑制されたことがわかりました。油、タンパク質、およびポリフェノールの大量のRNA単離を阻害します。我々は、RNAはシリカ膜に結合する工程の前に、溶解溶液でこれらの化合物を除去するために、RNA抽出過程を改変しました。重要な修飾は、1％のPVP（ステップ1.2）、遠心分離、溶解液からの上清の収集（1.7、1.8ステップ）の添加を含みました。 1％のPVPの添加の目的は、ポリフェノールを除去することです。我々が使用するPVPの最も効果的な濃度を検討し、1％のPVPがこのプロセスのための最も有効な濃度であることが確認されました。遠心分離した溶解液から上清を収集する目的油およびタンパク質を除去することです。上清は、上清中の油層とチューブ内のペレットを保持しないように注意してください収集する必要があります。これらの2つの簡単な修正が大幅に中央で種子や開発の後期段階（ 表1）からの回復、A260 / A280比、および単離されたRNAのA260 / A230比を改善しました。 標準プロトコルの簡単な変更を表して私たちの方法は、RNA単離のための市販のキットを使用しています。したがって、我々の方法は、塩化リチウムを使用し、フェノール - クロロホルム抽出、エタノール沈殿など煩雑な追加の工程を必要とせずに種子から高度に精製されたRNAの単離のために実用的です。私たちの方法は、修正が石油・タンパク質が豊富な作物の種子のために働くことを示す、また成熟した菜種や大豆の種子（ 表2）で使用するために有効です。 4 DAFの種子（ 表1）と若いルートからのRNA回収率秒、葉や茎は、我々の方法は、油やタンパク質に乏しい組織からのRNAの単離に適していないことを示す、我々の方法を使用して低かったです。したがって、この方法は、例えば、種子および果実などの油、タンパク質、及びポリフェノールを含有する組織からのRNAの単離のために有利です。 全体として、我々の方法は、RNAの精製のためのフェノール、クロロホルム、または追加のプロセスを使用せずに植物の種子から高度に精製されたRNAを取得するのに適しています。この方法は、種子における遺伝子発現のより正確なプロファイリングを容易にします。

植物は、次の世代を生じる種子を生産します。種子は、ポスト発芽成長のために、そのような油、炭水化物、およびタンパク質などの貯蔵物質、大量に蓄積します。人間は食品や動物飼料の供給源として種子貯蔵準備金を活用し、ひいては植物の種子は、世界的に食用有機物の主要なサプライヤの一つです。種子の収量を増加させると、植物科学における重要な課題です。 種子貯蔵物質が食品や工業材料の商業的に価値のある情報源であるため、これらの引当金の代謝の調節の根底にある分子メカニズムは、広く1-6を検討されています。さらに、これらのメカニズムを解明することは、作物種子収量を増加させるために有用であろう。種子は受精後植物卵巣で開発し、それらは発達段階1,6,7の一連の成熟します。さらに分子機構根底にある種子の開発を理解することは、詳細が必要です種子を開発する一連の正確な遺伝子発現プロファイルが生成されます。しかし、油、タンパク質、炭水化物、および植物種子中のポリフェノールの高い量は、それが困難な遺伝子発現の正確なプロファイリングを排除する高度に精製されたRNAを単離することを可能にします。 ここでは、油、タンパク質、およびポリフェノールを多く含む油糧種子からのRNAの単離のための効率的な方法を紹介します。この方法を使用して、研究者らは、高度に精製されたRNAを調製することができるであろう。このようなRNAは、開発および成熟脂肪種子における種子貯蔵物質の代謝調節を制御する重要な遺伝子で転写変化をモニタリングするために有用であろう。

<table border="0" cellpadding="0" cellspacing="0" width="655">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">RNeasy Plant Mini Kit</td>
			<td style="width:81px;">QIAGEN</td>
			<td style="width:136px;">74904</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">polyvinylpyrrolidone</td>
			<td style="width:81px;">Sigma-Aldrich</td>
			<td style="width:136px;">P5288-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">HOMOGENIZER S-303</td>
			<td style="width:81px;">AS ONE</td>
			<td style="width:136px;">1-1133-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">NanoDrop Lite</td>
			<td style="width:81px;">Thermo Scientific</td>
			<td style="width:136px;">ND-NDL-US-CAN</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">PrimeScript RT reagent Kit (Perfect Real Time)</td>
			<td style="width:81px;">TAKARA</td>
			<td style="width:136px;">RR037A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">KAPA SYBR Fast qPCR kit</td>
			<td style="width:81px;">Kapa biosystems</td>
			<td style="width:136px;">KK4601</td>
			<td></td>
		</tr>
	</tbody>
</table>

an efficient method for the isolation of highly purified rna from seeds for use in quantitative transcriptome analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

我々は最初のシロイヌナズナ完熟種子を用いて、PVPの最適濃度を調べました。全RNAを、0％、0.25％、0.5％、1.0％または2.0％のPVPを含有する細胞溶解緩衝液を用いて、上記のプロトコルに従って約1,000種から単離しました。油層と種子の破片（ 図1A）を回避しながら、ホモジナイズし、遠心分離後、上清を回収しました。 <img alt="図1" src="/files/ftp_upload/55008/55008fig1.jpg" /> 図1： 細胞溶解緩衝液中で最適なポリビニルピロリドン濃度の推定。総RNAは、PVPの異なる濃度を含有する細胞溶解緩衝液で抽出しました。 （A）ホモジナイズし、遠心分離後の種子の写真。 （B）各サンプルについて、波長の吸光度のグラフが示されています。<html> <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55008/55008fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 単離されたRNAの量及び純度は1.0％PVPを種子から精製したRNAを大量に単離する最も効果的な濃度であることが示された波長の吸光度のグラフ（ 図1B）から推定しました。 開発種子は実体顕微鏡（ 図2A、2B）の下で氷上で予冷したアルミニウム板上に、シロイヌナズナの果実から単離しました。種子は氷の上にアルミニウムラック（ 図2C、2D）にチューブ内のステンレス製の乳棒およびモーターグラインダーでホモジナイズしました。 2.JPG &quot;/&gt; 図2：単離と シロイヌナズナ の種子を開発するの均質化 。現像種子は、実体顕微鏡（A）下コールドアルミニウム板上果実から単離されます。心皮の茎（B）から剥離され、現像種子が収集されます。種子は、氷（D）で冷却アルミラックに試験管内のステンレス鋼乳棒（C）を用いて1％（w / v）のポリビニルピロリドンを含有する溶解緩衝液中で均質化されています。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55008/55008fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> 総RNAは、新しく開発された方法または従来の方法を使用して（以下、DAFと呼ばれる）開花後4,8、および12日目の種子から単離しました。濃度、A260 / A280比、およびA260 /200種から単離されたRNAのA230比を表1に示します。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>方法</td><td> DAF </td><td> RNA濃度</td><td> A260 / A280 </td><td> A260 / A230 </td></tr><tr><td rowspan="3">従来型の</td><td> 4 </td><td> 72.4±7.4 </td><td> 1.90±0.02 </td><td> 2.27±0.06 </td></tr><tr><td> 8 </td><td> 10.8±3.0 </td><td> 1.50±0.08 </td><td> 1.40±0.17 </td></tr><tr><td> 12 </td><td> 4.9±1.7 </td><td> 1.57±0.10 </td><td> 0.76±0.18 </td></tr><tr><td rowspan="3">新しい</td><td> 4 </td><td> 63.7±2.6 </td><td> 2.10±0.08 </td><td> 2.29±0.03 </td></tr><tr><td> 8 </td><td> 46.3±4.9 </td><td> 2.15±0.05 </td><td> 2.23±0.09 </td></tr><tr><td> 12 </td><td> 41.77; 3.1 </td><td> 2.09±0.04 </td><td> 2.17±0.07 </td></tr></tbody></table> 表1： シロイヌナズナの 種子 から単離されたRNAの濃度、A260 / A280比、およびA260 / A230比 。全RNAは、従来の方法または新しく開発された方法を用いて、8、4時（190〜206種子から）約200種子から単離し、12 DAFました。 RNA濃度は、A260 / A280比、およびA260 / A230比を測定しました。値は、3つの独立した実験の平均±SDを表します。 結果は、我々の方法は、8および12 DAF種子から高度に精製されたRNAを分離することができたことを示しています。全RNAをcDNAに逆転写した、およびcDNA溶液を100倍に希釈しました。コピーWRI1の番号（ しわ1：AT3G54320）、SDP1（SUGAR DEPENDENT1：AT5G04040）7 &lt;/ SUP&gt;、及びEIF3K（真核生物翻訳開始因子3K：AT4G33250）内部統制8とを測定し、転写物の相対量は、（ 図3）分析、定量的リアルタイムPCRによって評価しました。 <img alt="図3" src="/files/ftp_upload/55008/55008fig3.jpg" /> 図3： 種子の開発に低レベルの転写物の定量。全RNAを、我々の方法を用いて、4、8、及び12 DAF種子から抽出し、そしてcDNA溶液を調製しました。 WRI1（A）及びSDP1（B）転写物の相対量は、定量的リアルタイムPCRにより測定しました。 EIF3K内部対照として使用しました。値は3回の独立した実験の平均±SDを表します。 DAF;開花後の日。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/55008/55008fig3large.jpg" target="_blank">彼をクリックしてください</a>この図の拡大版を表示するために再。 WRI1とSDP1転写物の量は、種子の開発に比較的低いが、異なる発達段階での種子の間の発現の変化を検出することが可能でした。私たちの方法は、他の油糧種子で利用することができることを確認するには、全RNAは、 アブラナおよびダイズの成熟種子から単離されました。 RNA濃度は、A260 / A280比、およびA260 / A230比を表2に示します。 <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td>方法</td><td>工場</td><td> RNA濃度</td><td> A260 / A280 </td><td> A260 / A230 </td></tr><tr><td>従来型の</td><td>セイヨウアブラナ</td><td> 7.7±0.8 </td><td> 1.5±0.12 </td><td> 0.75±0.11 </td></tr><tr><td></td><td> G. maxの</td><td&gt; 9.7±2.3 </td><td> 1.61±0.06 </td><td> 0.69±0.09 </td></tr><tr><td>新しい</td><td>セイヨウアブラナ</td><td> 143.2±19.0 </td><td> 2.17±0.03 </td><td> 2.23±0.13 </td></tr><tr><td></td><td> G. maxの</td><td> 152.3±4.4 </td><td> 2.17±0.02 </td><td> 2.30±0.02 </td></tr></tbody></table> 表2：濃度、A260 / A280比、および セイヨウアブラナ および ダイズ の成熟種子から単離されたRNAのA260 / A230比 。全RNAは、従来の方法または新しく開発された方法を用いて、 アブラナおよびダイズ種子の約100 mgを単離しました。種子を液体窒素中で冷却し、乳鉢と乳棒を用いて以前に粉砕し、概ね均質化しました。 RNA濃度、A260 / A280の比率、およびA260 / A230比を測定しました。値は、3つの独立した実験の平均±SDを表します。

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An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

定量的トランスクリプトーム解析に使用するための種子から高度に精製されたRNAの単離のための効率的な方法

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

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Nanyang Technological University

Research

JoVE Journal

Biochemistry

9.6K ビュー.  National Institute for Basic Biology. We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

ビデオ: An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

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

Experimental Design for Laser Microdissection RNA-Seq: Lessons from an Analysis of Maize Leaf Development

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not detected or are not identified as differentially expressed (DE) in transcriptomic analyses of whole plant organs. Laser Microdissection RNA-Seq (LM RNA-Seq) is a powerful tool to identify genes that are DE in specific developmental domains. However, the choice of cellular domains to microdissect and compare, and the accuracy of the microdissections are crucial to the success of the experiments. Here, two examples illustrate design considerations for transcriptomics experiments; a LM RNA-seq analysis to identify genes that are DE along the maize leaf proximal-distal axis, and a second experiment to identify genes that are DE in liguleless1-R (lg1-R) mutants compared to wild-type. Key elements that contributed to the success of these experiments were detailed histological and in situ hybridization analyses of the region to be analyzed, selection of leaf primordia at equivalent developmental stages, the use of morphological landmarks to select regions for microdissection, and microdissection of precisely measured domains. This paper provides a detailed protocol for the analysis of developmental domains by LM RNA-Seq. The data presented here illustrate how the region selected for microdissection will affect the results obtained.

Many developmentally important genes have cell- or tissue-specific expression patterns. This paper describes LM RNA-seq experiments to identify genes that are differentially expressed at the maize leaf blade-sheath boundary and in lg1-R mutants compared to wild-type. The experimental considerations discussed here apply to transcriptomic analyses of other developmental phenomena.

レーザーマイクロダイセクションRNA-のSeqのための実験デザイン：トウモロコシ葉の開発の分析からの教訓

Genes with important roles in development frequently have spatially and/or temporally restricted expression patterns. Often these gene transcripts are not ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

定量的質量分析のための脂肪滴の分離

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Highly Sensitive and Quantitative Detection of Proteins and Their Isoforms by Capillary Isoelectric Focusing Method

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides an alternative strategy, capillary isoelectric focusing (cIEF), with many advantages compared to conventional immunoblotting. This is an antibody-based, automated, rapid, and quantitative method in which a complete western blotting procedure takes place inside an ultrathin capillary. This technique does not require a gel to transfer to a membrane, stripping of blots, or x-ray films, which are typically required for conventional immunoblotting. Here, proteins are separated according to their charge (isoelectric point; pI), using less than a microliter (400 nL) of total protein lysate. After electrophoresis, proteins are immobilized onto the capillary walls by ultraviolet light treatment, followed by primary and secondary (horseradish peroxidase (HRP) conjugated) antibody incubation, whose binding is detected through enhanced chemiluminescence (ECL), generating a light signal that can be captured and recorded by a charge-coupled device (CCD) camera. The digital image can be analyzed and quantified (peak area) using software. This high throughput procedure can handle 96 samples at a time; is highly sensitive, with protein detection in the picogram range; and produces highly reproducible results because of automation. All of these aspects are extremely valuable when the quantity of samples (e.g., tissue samples and biopsies) is a limiting factor. The technique has wider applications as well, including screening of drugs or antibodies, biomarker discovery, and diagnostic purposes.

Capillary isoelectric focusing is an antibody-based, ultrasensitive, high throughput technique, enabling detailed characterization of proteins and their isoforms from extremely small biological samples. The following describes a protocol for detection and quantification of specific proteins and their isoforms in an automated and robotized manner.

タンパク質キャピラリー等電点焦点法によるアイソ フォームの高感度・定量的検出

Immunoblotting has become a routine technique in many laboratories for protein characterization from biological samples. The following protocol provides ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

植物の鉄含有量を測定するための比色定量法

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Toeprinting Analysis of Translation Initiation Complex Formation on Mammalian mRNAs

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation of protein synthesis is the key to cellular stress-response, and dysregulation is central to many disease states, such as cancer. For instance, although cellular stress leads to the inhibition of global translation by attenuating cap-dependent initiation, certain stress-response proteins are selectively translated in a cap-independent manner. Discreet RNA regulatory elements, such as cellular internal ribosome entry sites (IRESes), allow for the translation of these specific mRNAs. Identification of such mRNAs, and the characterization of their regulatory mechanisms, have been a key area in molecular biology. Toeprinting is a method for the study of RNA structure and function as it pertains to translation initiation. The goal of toeprinting is to assess the ability of in vitro transcribed RNA to form stable complexes with ribosomes under a variety of conditions, in order to determine which sequences, structural elements, or accessory factors are involved in ribosome binding&#8212;a pre-cursor for efficient translation initiation. Alongside other techniques, such as western analysis and polysome profiling, toeprinting allows for a robust characterization of mechanisms for the regulation of translation initiation.

Toeprinting aims to measure the ability of in vitro transcribed RNA to form translation initiation complexes with ribosomes under a variety of conditions. This protocol describes a method for toeprinting mammalian RNA and can be used to study both cap-dependent and IRES-driven translation.

哺乳類の Mrna の翻訳開始複合体形成の Toeprinting 分析

Translation initiation is the rate-limiting step of protein synthesis and represents a key point at which cells regulate their protein output. Regulation ...

Rapid Isolation of the Mitoribosome from HEK Cells

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative phosphorylation complexes encoded by the mitochondrial genome. Since all proteins synthesized by human mitoribosomes are integral membrane proteins, human mitoribosomes are tethered to the mitochondrial inner membrane during translation. Compared to the cytosolic ribosome the mitoribosome has a sedimentation coefficient of 55S, half the rRNA content, no 5S rRNA and 36 additional proteins. Therefore, a higher protein-to-RNA ratio and an atypical structure make the human mitoribosome substantially distinct from its cytosolic counterpart.
Despite the central importance of the mitoribosome to life, no protocols were available to purify the intact complex from human cell lines. Traditionally, mitoribosomes were isolated from mitochondria-rich animal tissues that required kilograms of starting material. We reasoned that mitochondria in dividing HEK293-derived human cells grown in nutrient-rich expression medium would have an active mitochondrial translation, and, therefore, could be a suitable source of material for the structural and biochemical studies of the mitoribosome. To investigate its structure, we developed a protocol for large-scale purification of intact mitoribosomes from HEK cells. Herein, we introduce nitrogen cavitation method as a faster, less labor-intensive and more efficient alternative to traditional mechanical shear-based methods for cell lysis. This resulted in preparations of the mitoribosome that allowed for its structural determination to high resolution, revealing the composition of the intact human mitoribosome and its assembly intermediates. Here, we follow up on this work and present an optimized and more cost-effective method requiring only ~1010 cultured HEK cells. The method can be employed to purify human mitoribosomal translating complexes, mutants, quality control assemblies and mitoribosomal subunits intermediates. The purification can be linearly scaled up tenfold if needed, and also applied to other types of cells.

Mitochondria have specialized ribosomes that diverged from their bacterial and cytoplasmic counterparts. Here we show how mitoribosomes can be obtained from their native compartment in HEK cells. The method involves isolation of mitochondria from suspension cells and consequent purification of mitoribosomes.

HEK 細胞から Mitoribosome の急速な隔離

The human mitochondria possess a dedicated set of ribosomes (mitoribosomes) that translate 13 essential protein components of the oxidative ...

Translating Ribosome Affinity Purification (TRAP) for RNA Isolation from Endothelial Cells In Vivo

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis of transcriptome and gene expression by qPCR and RNA sequencing. Comprehensive transcriptome and gene expression analysis of specific cell types in complex tissues and organs will be critical to understand cellular and molecular mechanisms by which genes are regulated and their association with tissue homeostasis and organ functions. In this article, we demonstrate the methodology for isolation of ribosome-bound RNA directly in vivo in the vascular endothelia of animal lungs as an example. The specific materials and procedures for tissue processing and RNA purification will be described, including the assessment of RNA quality and yield as well as real time qPCR for arteriogenic gene assays. This approach, known as translating ribosome affinity purification (TRAP) technique, can be utilized for characterization of gene expression and transcriptome analysis of certain cell types directly in vivo in any specific type in complex tissues.

Translating Ribosome Affinity Purification TRAP for RNA Isolation from Endothelial Cells In Vivo

We present an approach to purify ribosome-bound mRNA from vascular endothelial cells (ECs) directly in mouse brain, lung and heart tissues via EC-specific genetic tag of enhanced green fluorescence protein (EGFP)in ribosomes in combination with RNA purification.

生体内細胞からのRNA単離に対するリボソーム親和性精製(TRAP)の翻訳

Many studies have been limited to using in vitro cellular assays and whole tissues or isolating of specific cell types from animals for in vitro analysis ...

Obtaining High-Quality Transcriptome Data from Cereal Seeds by a Modified Method for Gene Expression Profiling

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality RNA is often hindered by high starch and sugar content. These compounds can reduce both the yield and the quality of the extracted total RNA. The deterioration in quantity and quality of total RNA can subsequently have a significant impact on the downstream transcriptomic analyses, which may not accurately reflect the spatial and/or temporal variation in the gene expression profile of the samples being tested. In this protocol, we describe an optimized method for extraction of total RNA with sufficient quantity and quality to be used for whole transcriptome analysis of cereal grains. The described method is suitable for several downstream applications used for transcriptomic profiling of developing, germinating, and mature cereal seeds. The method of transcriptome profiling using a microarray platform is shown. This method is specifically designed for gene expression profiling of cereals with described genome sequences. The detailed procedure from microarray handling to final quality control is described. This includes cDNA synthesis, cRNA labelling, microarray hybridization, slide scanning, feature extraction, and data quality validation. The data generated by this method can be used to characterize the transcriptome of cereals during germination, in various stages of grain development, or at different biotic or abiotic stress conditions. The results presented here exemplify high-quality transcriptome data amenable for downstream bioinformatics analyses, such as the determination of differentially expressed genes (DEGs), characterisation of gene regulatory networks, and conducting transcriptome-wide association study (TWAS).

A method for transcriptome profiling of cereals is presented. The microarray-based gene expression profiling starts with the isolation of high-quality total RNA from cereal grains and continues with the generation of cDNA. After cRNA labelling and microarray hybridization, recommendations are given for signal detection and quality control.

遺伝子発現プロファイリングのための修飾法による穀物種子からの高品質なトランスクリプトームデータの取得

The characterization of gene expression is dependent on RNA quality. In germinating, developing and mature cereal seeds, the extraction of high-quality ...

Use of Microscale Thermophoresis to Measure Protein-Lipid Interactions

The ability to determine the binding affinity of lipids to proteins is an essential part of understanding protein-lipid interactions in membrane trafficking, signal transduction and cytoskeletal remodeling. Classic tools for measuring such interactions include surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). While powerful tools, these approaches have setbacks. ITC requires large amounts of purified protein as well as lipids, which can be costly and difficult to produce. Furthermore, ITC as well as SPR are very time consuming, which could add significantly to the cost of performing these experiments. One way to bypass these restrictions is to use the relatively new technique of microscale thermophoresis (MST). MST is fast and cost effective using small amounts of sample to obtain a saturation curve for a given binding event. There currently are two types of MST systems available. One type of MST requires labeling with a fluorophore in the blue or red spectrum. The second system relies on the intrinsic fluorescence of aromatic amino acids in the UV range. Both systems detect the movement of molecules in response to localized induction of heat from an infrared laser. Each approach has its advantages and disadvantages. Label-free MST can use untagged native proteins; however, many analytes, including pharmaceuticals, fluoresce in the UV range, which can interfere with determination of accurate KD values. In comparison, labeled MST allows for a greater diversity of measurable pairwise interactions utilizing fluorescently labeled probes attached to ligands with measurable absorbances in the visible range as opposed to UV, limiting the potential for interfering signals from analytes.

Microscale thermophoresis obtains binding constants quickly at low material cost. Either labeled or label free microscale thermophoresis is commercially available; however, label free thermophoresis is not capable of the diversity of interaction measurements that can be performed using fluorescent labels. We provide a protocol for labeled thermophoresis measurements.