本研究は台湾 (省の科学と技術、最も 100-2923-B-002-002-MY3、H.J.L.、106-2313-B-002-011-MY3) からの助成金によって支えられたチェコ共和国 (南ボヘミア州庁大学 GAJU 付与 065/2017/P Y.H.L) 付与、およびスペイン (スペイン経済省と競争力、CGL2012 36251 と CGL2015 64727 P コドーピング、およびカタロニアの政府への許可を付与 2014 SGR 619 コドーピングに);それはまた経済と地域開発 (コドーピング フェダーイン ・資金) の欧州基金から助成を受けた。

1 合成と dsRNA の準備
<ol><li>標的遺伝子の 3' 非翻訳領域の dsRNA のターゲット サイトを識別します。Α-チューブリン (浴槽) 遺伝子をターゲットに、dsTub を使用 (GenBank 受入番号: KX228233) と dsEGFP のシーケンスから否定的な dsRNA のコントロールは、強化された緑色蛍光タンパク質 (EGFP;GenBank の受入番号: LC311024)。</li>
	<li>T7 プロモーター配列を含む遺伝子特定のプライマーで dsRNA テンプレートを合成する標準 PCR 増幅を実行 (5'-TAATACGACTCACTATAGGG-3')。PCR 増幅条件と使用されるプライマー情報は、林らによってそれらで報告されました。(2017)12 (材料の表を参照してください)。</li>
	<li>T7 転写を用いた dsRNA 準備を進めるキット (材料の表を参照してください)、製造元の指示に従います。 注:高数量 dsRNA、(合計 40 μ L) の 1 つの管の 2 つの反応をミックスし、37 ° C で一晩インキュベートします。</li>
	<li>DNase の 2 μ L を追加 (材料の表を参照してください) に dsRNA サンプル ダイジェスト DNA テンプレートを 37 ° C で 15 分間。</li>
	<li>200 μ L の抽出試薬を追加 (材料表参照) 40 μ L のクロロホルムし渦。</li>
	<li>4 ° C で 13,000 x g で 10 分間遠心し、新しい微量遠心チューブに上清 (150 μ L) を収集します。</li>
	<li>150 μ L のイソプロパノールを追加し、氷で 15 分間インキュベート dsRNA を沈殿させます。</li>
	<li>4 ° C で 13,000 × g で 15 分間遠心し、上清を除去します。</li>
	<li>DsRNA ペレットを洗浄する 70% エタノール 200 μ L を追加します。4 ° C で 13,000 x g で 10 分間遠心</li>
	<li>エタノールを削除し、エタノールの洗浄ステップを繰り返します。</li>
	<li>エタノールと、3 分間遠心濃縮、乾燥 dsRNA ペレットを削除し、RNase フリー水で dsRNA を再懸濁します。</li>
	<li>マイクロ ボリュームが紫外可視分光光度計を使用して精製された dsRNA の数量を決定 (材料表参照) 製造元の指示に従って、必要な濃度 (例えばの準備のための 0.25 μ g/μ L に調整dsRNA リポプレックス)。</li>
	<li>3 ヵ月の-20 ° C で dsRNA のサンプルを格納します。</li>
</ol>2. dsRNA リポプレックスの準備
<ol><li>トランスフェクション試薬の 4 μ L を希釈 (材料表参照) 5% ブドウ糖溶液の 4 μ L を追加することによって。5% ブドウ糖液の 4 μ L を追加することで (1 μ g) dsRNA の 4 μ L を希釈します。</li>
	<li>希釈したリポソーム試薬溶液すぐに希釈された dsRNA のソリューションにすべて一度に追加します。簡単に、それらをミックスし、25 ° C で 15 分間インキュベートする渦 注:逆の順序でソリューションを混在させないでください。</li>
	<li>DsRNA リポプレックス使用の準備ができています。準備の 1 時間内で使用し、1 時間後に破棄します。 注:製造元の指示に従って、リポプレックスをできるだけ早く使用必要があります。</li>
</ol>3. 体液や腸ジュースから菌体外酵素のコレクション
<ol><li>
昆虫生理食塩水バッファー (10 x の在庫)
	<ol>
<li>109.3 g 塩化ナトリウム、15.7 g KCl、6.3 g CaCl2、および 8.3 g MgCl2·6H2O. 二重蒸留水 900 mL をミックスします。</li>
		<li>6.8、pH 調整、1,000 mL までを記入してください。</li>
	</ol>
</li>
	<li>
体液コレクション
	<ol>
<li>3 分の移動しないまで氷にゴキブリを麻酔します。</li>
		<li>2 本の指 (親指と人差し指) でゴキブリを押しながらゴキブリの腹側を上げます。</li>
		<li>解剖はさみを使用して、後ろ足の coxa の先端をカットします。 注:Coxa と転子間の接続交差点をカットします。Coxa の他の部分の切除は体液を収集するために効率が低下します。</li>
		<li>中指で軽く腹部を圧迫し、10 μ L マイクロ ピペットを使用して切開から出血体液を収集します。</li>
		<li>微量遠心チューブにいくつかゴキブリ (5 人) から体液をプールします。 注:マイクロ遠心チューブ用メラニン化を防ぐために氷の上を保ちます。男性のゴキブリから得られた体液量の平均は 2-3 μ L です。</li>
		<li>10、ベンチトップ遠心分離と簡単に体液をスピン s。</li>
		<li>(すなわち、10 μ L)、体液の適切なボリュームを転送し、1 x 昆虫生理食塩水バッファーの 50 μ L でそれをミックスします。</li>
		<li>血球スピンダウンに 4 ° C で 1,000 x g で 10 分間遠心します。きれいな遠心チューブに上清を転送します。</li>
		<li>A28015を測定することにより微量紫外可視分光光度計を使用して総蛋白質濃度を定量化します。</li>
		<li>同じタンパク質濃度 (6 mg/μ L の総蛋白) を持っている各サンプルを調整します。</li>
	</ol>
</li>
	<li>
腸ジュース コレクション
	<ol>
<li>氷の上のゴキブリを麻酔します。ゴキブリ腹側解剖皿の上で固定冷たい 1 x 昆虫生理食塩水バッファー昆虫ピンを使用します。</li>
		<li>高級ピンセットで腹部を解剖します。昆虫生理食塩水バッファー × 1 新鮮な料理に全体腸 (フロント、中間、および後肢腸を含む) を削除します。</li>
		<li>前部および後ろ足の腸を削除し、100 μ L 昆虫生理食塩水バッファーを含む微量遠心チューブに腸をすばやく転送します。</li>
		<li>同じ管で 10 の渦 6 ゴキブリから蚊をプール s。</li>
		<li>スピン ・ ダウン血液細胞と腸の組織に 4 ° C で 1,000 x g で 10 分間遠心します。きれいな遠心チューブに上清を転送します。</li>
		<li>A28015を測定することにより微量紫外可視分光光度計を使用して総蛋白質濃度を定量化します。</li>
		<li>同じタンパク質濃度 (6 mg/μ L の総蛋白) を持っている各サンプルを調整します。</li>
	</ol>
</li>
</ol>4. dsRNA 劣化分析
<ol><li>新鮮な裸の dsRNA の dsRNA リポプレックス (1 μ g) 4 μ L を準備します。</li>
	<li>体液、または腸から dsRNA 抽出ソリューション昆虫生理食塩水バッファー (コントロール) の 10 μ L の酵素ジュースをミックスします。</li>
	<li>2 μ L の酵素活性を阻害するコントロールとして試料を分離するグリコールエーテルジアミン四酢酸 (20 mM) または RNase フリー水のいずれかを追加します。</li>
	<li>1 時間 (6、12、または 24 時間以上) 25 ° C でインキュベートします。</li>
	<li>200 μ L の抽出試薬と 40 μ L のクロロホルム、渦、追加します。</li>
	<li>4 ° C で 13,000 x g で 10 分間遠心し、新しい微量遠心チューブに上清 (150 μ L) を収集します。</li>
	<li>150 μ L のイソプロパノールを追加し、氷で 15 分間インキュベート dsRNA を沈殿させます。</li>
	<li>4 ° C で 13,000 × g で 15 分間遠心し、上清を除去します。</li>
	<li>DsRNA ペレットを洗浄する 70% エタノール 200 μ L を追加します。4 ° C で 13,000 x g で 10 分間遠心</li>
	<li>エタノールを削除し、エタノールの洗浄ステップを繰り返します。</li>
	<li>エタノール、10 μ L の RNase フリーの水で 3 分再懸濁します dsRNA の遠心濃縮、乾燥 dsRNA ペレットを削除します。</li>
	<li>1.5% の agarose のゲルの電気泳動によって扱われた dsRNA の整合性をチェックします。</li>
</ol>5. dsRNA の内服
<ol><li>ドライフード (犬の食事) の自由食事にゴキブリを維持します。DsRNA 摂取実験の前日、ゴキブリから水の供給を奪います。</li>
	<li>フレキシブル鉗子 (図 1、ステップ 5) でその翼を掴んでゴキブリを保持します。 注:ゴキブリの体の部分をつかむか。</li>
	<li>セクション 2 の説明に従って、フィード dsRNA リポプレックスとして裸の dsRNA を準備します。 注:前に餌、dsRNA リポプレックスをたて準備されるべき。</li>
	<li>フィードの dsRNA リポプレックスまたは裸の dsRNA の 4 μ L (250 ng) マイクロ ピペットで。</li>
	<li>ゆっくりピペット dsRNA ソリューションの液滴、ゴキブリの口器に近いし、ゴキブリは完全に液滴を摂取します。</li>
	<li>8 日か 16 日間連続 (1 h 点灯後、消灯前に 1 h) は 1 日 2 回の摂取実験を実行します。 注:ゴキブリは水だけ摂取実験中に dsRNA のソリューションまたはコントロール試薬 (例えば、ヌクレアーゼ フリー水、ブドウ糖溶液、リポソーム試薬) とマニュアル給餌を取得しました。</li>
	<li>ゴキブリに dsRNA 実験の摂取の 16 日後の水のボトルを提供します。</li>
</ol>6. ノックダウンを評価します。
<ol><li>選択した時にポイント (dsRNA 摂取試験後 2、9、17 日) は dsEGFP と dsTub 扱われる昆虫を収集そして選ばれた組織 (例えば腸) を解剖します。</li>
	<li>量的なリアルタイム PCR (qRT PCR) を実行します。QRT PCR 増幅条件およびプライマー情報林らによって報告されました。(2017)12。</li>
	<li>コントロールと死亡を確認し、移動は、もはやゴキブリを削除は毎日昆虫 dsRNA 治療を評価します。</li>
</ol>

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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advances+in+lipid+nanoparticles+for+siRNA+delivery.">Advances in lipid nanoparticles for siRNA delivery.</a> Pharmaceutics. 5, 498-507 (2013).</li><li>Xia, Y., Tian, J., Chen, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+surface+properties+on+liposomal+siRNA+delivery.">Effect of surface properties on liposomal siRNA delivery.</a> Biomaterials. 79, 56-68 (2016).</li></ol>

著者が明らかに何もありません。

このプロトコルは、ドイツのゴキブリの中腸ジュースのリボヌクレアーゼ消化に対する保護を含む dsRNA リポプレックスの口頭配達を通じて効果的な RNAi のメソッドを示します。様々 な昆虫の種の他の研究のように、dsRNA8,9,10の劣化による dsRNA の口頭配達を通じて貧しい RNAi 効果がほとんどを占めています。このプロトコ?...

RNAi は、多くの真核生物1の dsRNA の分子によってトリガーされる転写後サイレンシング経路の機構を通じてノックダウン遺伝子発現する効果的な方法として実証されています。過去の研究では、RNAi オゾン注入を介して特定の遺伝子発現および/または昆虫2,3のさまざまな分類群の dsRNA の供給によって動作する開発から遺伝子の機能を研究するための有用なツールとなっています。特異性と破壊効果の頑健性のため、RNAi のアプリケーションは現在害虫制御管理4、5のための潜在的な戦略として検討されています。ただし、RNAi の効率は昆虫種間対象別の遺伝子と配信方法によって異なります。証拠の成長するボディは、リボヌクレアーゼによる劣化は、dsRNA の不安定性 RNAi5,6の限られた有効性の重要な要因であることを示唆しています。例えば、 Manduca sextaの RNAi の感度が低いは、体液混合 dsRNA が 1 時間7内ですぐに劣化したという事実によって説明されています。同様に、効率的に摂取された dsRNA が低下する、中腸でアルカリ性の核酸分解酵素の存在の異なる昆虫8,9,10で RNAi 効率が低い相関が強い。
DsRNA の口頭配達は害虫制御戦略の RNAi のアプリケーションの特に興味深いですが中腸における核酸による dsRNA の劣化を遅らせる方法はまだ開発されていない、有効なことを確認する可能性のあるだろうRNAi を介して供給します。ただし、dsRNA、カイコ、例えば50 μ g の大量の餌やイナゴ種で 8 日間 (合計 8 dsRNA μ g)、継続的に供給で dsRNA の口頭配達する RNAi の応答が報告されています。チャバネゴキブリ germinicaチャバネゴキブリが dsRNA11,12,13,14、注入による RNAi に敏感を介して供給 dsRNA に敏感ではないです。最近では、林ら(2017) は dsRNA カプセルこと成功した rnai ノックダウンするリポソーム キャリア結果とα-チューブリン遺伝子発現15チャバネゴキブリの中腸とトリガー有意な死亡率を示しています。中腸における dsRNA の劣化は、口腔の RNAi のための制限要因は、リポソーム キャリア劣化は、腸内で強いヌクレアーゼ活動とその他の昆虫に適用が容易ですから dsRNA を保護する手段として役割を果たします。注特定のトランスフェクション試薬を選ぶ理由の現在のプロトコルのリポソーム キャリアはそれは毒性が少なく、昆虫の細胞ラインの transfection のためテストされていますを使いました (材料の表を参照) によると、節してください。Gharaviらの異なるリポソーム トランスフェクション システムとの比較によって(2013)16、株小さい干渉の RNA (siRNA) の効率はおよそこれと他の昆虫の17,18 dsRNA 配信システムに使用されている他の市販システム間で同じ.さらに、私たちの供給法は各ゴキブリで dsRNA の適切な量を摂取し、結果が得られる堅牢で確認をように十分注意してください。要約すると、議定書と結果を示す、dsRNA リポプレックスを使用して dsRNA の安定性を向上させる将来的に害虫駆除のための有望なアプローチである、RNAi の戦略の口頭配達のデザインへの扉を開きます。

<table border="0" cellpadding="0" cellspacing="0" style="width:1124px;" width="1123">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">GenJe Plus DNA in vitro Transfection reagent</td>
			<td style="width:133px;">SignaGen</td>
			<td style="width:157px;">SL100499</td>
			<td style="width:481px;">for lipoplexes preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Blend Taq plus</td>
			<td style="width:133px;">TOYOBO&#160;</td>
			<td style="width:157px;">BTQ-201</td>
			<td>for PCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast SYBR Green Master Mix</td>
			<td style="width:133px;">ABI&#160;</td>
			<td style="width:157px;">4385612</td>
			<td>for qPCR</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">FirstChoice RLM-RACE Kit</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">AM1700</td>
			<td>for 3&#39; UTR identification&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">MEGAscript T7 Transcription Kit</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">AMB13345</td>
			<td>for dsRNA synthesis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TURBO DNase</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">AM2239</td>
			<td>remove DNA template from dsRNA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIzol</td>
			<td style="width:133px;">Invitrogen</td>
			<td style="width:157px;">15596018</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">RQ1 RNase-Free Dnase</td>
			<td style="width:133px;">Promega</td>
			<td style="width:157px;">M6101</td>
			<td>remove DNA template from total RNA&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chloroform&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">C2432</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">2-Propanol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">I9516</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ethanol</td>
			<td>Sigma-Aldrich</td>
			<td>24102</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Diethyl pyrocarbonate, DEPC</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">D5758</td>
			<td>for RNase free water preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">glucose solution</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">G3285</td>
			<td>for lipoplexes preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride, NaCl</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">S7653</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">Potassium chloride, KCl</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">P9333</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:352px;">Calcium chloride, CaCl2</td>
			<td>Sigma-Aldrich</td>
			<td>C1016</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;">Magnesium chloride hexahydrate, MgCl2.6H2O</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">M2670</td>
			<td>insect saline buffer formula</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">EGTA&#160;</td>
			<td>Sigma-Aldrich</td>
			<td style="width:157px;">E3889</td>
			<td>enzyme inhibitor&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">dissecting scissor</td>
			<td style="width:133px;">F.S.T.</td>
			<td style="width:157px;"></td>
			<td>cockroach dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fine tweezers</td>
			<td style="width:133px;">F.S.T.</td>
			<td style="width:157px;"></td>
			<td>cockroach dissection</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">flexible tweezer</td>
			<td style="width:133px;">F.S.T.</td>
			<td style="width:157px;"></td>
			<td>cockroach holding&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pipetman</td>
			<td style="width:133px;">RAININ</td>
			<td style="width:157px;">P10</td>
			<td>sample preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">microcentrifuge tube</td>
			<td style="width:133px;">Axygen</td>
			<td>MCT175C, PCR02C</td>
			<td>sample preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">pipette tip&#160;</td>
			<td style="width:133px;">Axygen</td>
			<td></td>
			<td>sample preparation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">vortexter</td>
			<td style="width:133px;">Digisystem</td>
			<td style="width:157px;">vm1000</td>
			<td>sample preparation</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">Minispin centrifuge</td>
			<td style="width:133px;">The Gruffin Group</td>
			<td style="width:157px;">GMC 206</td>
			<td>for liquid spin down&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:133px;">ALC</td>
			<td style="width:157px;">PK121R</td>
			<td>sample preparation</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">pH meter&#160;</td>
			<td style="width:133px;">JENCO</td>
			<td style="width:157px;">6071</td>
			<td>for pH adjust</td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;">micro-volume spectrophotometer</td>
			<td style="width:133px;">Quawell</td>
			<td style="width:157px;">Q3000</td>
			<td>nucleic acid quantitative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">PCR Thermal cycler</td>
			<td style="width:133px;">ABI&#160;</td>
			<td style="width:157px;">2720</td>
			<td>for template PCR or&#160; dsRNA synthesis incubation&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">quantitative real-time PCR</td>
			<td style="width:133px;">ABI&#160;</td>
			<td style="width:157px;">StepOne plus</td>
			<td>gene expression quantitative</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifugal Vacuum Concentrators</td>
			<td style="width:133px;">eppendorf</td>
			<td style="width:157px;">5301</td>
			<td>for dsRNA or total RNA extraction</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Multipette&#160;</td>
			<td style="width:133px;">eppendorf</td>
			<td style="width:157px;">xstream</td>
			<td>for real-time PCR sample loading&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Agarose I</td>
			<td style="width:133px;">amresco</td>
			<td style="width:157px;">0710</td>
			<td>for nucleic acid electrophoresis</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub gene specfifc forward preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>GGG ACA AGC CGG AGT GCA GA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub gene specfifc reverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TCC TGC TCC TGT CTC GCT GA</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsTub template forward primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGA CAA GCC GGA GTG CAG&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsTub template reverse primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGT CCT GCT CCT GTC TCG CTG&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsEGFP template forward preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGT ATG GTG AGC AAG GGC GAG GAG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">dsEGFP template reverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>TAA TAC GAC TCA CTA TAG GGT GGC GGA TCT TGA AGT TCA CC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub qPCR forward primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>GGA CCG CAT CAG GAA ACT GGC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">tub qPCR reverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>CCA CAG ACA GCC TCT CCA TGA GC</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">ef1 qPCR forward primer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>CGC TTG AGG AAA TCA AGA AGG A</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:352px;">ef1 qPCRreverse preimer</td>
			<td style="width:133px;">tri-I biotech</td>
			<td style="width:157px;"></td>
			<td>CCT GCA GAG GAA GAC GAA G</td>
		</tr>
	</tbody>
</table>

practical use of rna interference: oral delivery of double-stranded rna in liposome carriers for cockroaches

RNA 干渉 (RNAi) 多数の遺伝子の生物学的機能を暴くため広く用いられてきた、重要な遺伝子発現の中断動作害虫コントロール ツールとして想定されました。二本鎖 RNA (dsRNA) を正常に配信のインジェクション、給餌と、浸漬など、さまざまな方法が報告されている dsRNA の経口デリバリーによる RNAi の効率は非常に異なる昆虫グループ間で可変。チャバネゴキブリチャバネゴキブリは、以前発表された多くの研究で示されている dsRNA の注入に非常に敏感。本研究では、リポソーム キャリアとカプセル化された dsRNA が腸ジュースで dsRNA の劣化を遅らせるに十分であることを示す方法について説明します。とりわけ、大幅リポソームによってカプセル化された dsRNA の連続的供給、中腸におけるチューブリンの発現を減少させる、ゴキブリの死をもたらした。結論としては、定式化と核酸に対して dsRNA を保護する dsRNA リポプレックスの利用かもしれない害虫制御のための RNAi の実用的な使用、将来的に。

DsRNA の経口配信用プロトコルの簡易方式はで示した図 1、dsRNA リポプレックスの準備のため、主要な手順が表示されます。
チャバネゴキブリ、dsTub リポプレックス腸ジュースで培養を行った前のヴィヴォアッセイおよび dsRNA の整合性の中腸ジュースの dsRNA の劣化にリポソーム キャリア...

Watch this Scientific Journal Video about Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches at JoVE.com

Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

RNA 干渉法の活用: ゴキブリのリポソーム キャリアで二本鎖 RNA の経口デリバリー

この原稿は、リポソームにカプセル化された二本鎖 RNA の経口摂取によってドイツのゴキブリの中腸における遺伝子発現の減少を示しています。

RNA interference (RNAi) has been widely applied for uncovering the biological functions of numerous genes, and has been envisaged as a pest control tool ...

practical-use-rna-interference-oral-delivery-double-stranded-rna

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

9.3K ビュー.  National Taiwan University. この原稿は、リポソームにカプセル化された二本鎖 RNA の経口摂取によってドイツのゴキブリの中腸における遺伝子発現の減少を示しています。

ビデオ: Practical Use of RNA Interference: Oral Delivery of Double-stranded RNA in Liposome Carriers for Cockroaches

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

HeLa細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

生物学

Chromatin Isolation by RNA Purification (ChIRP)

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental gene expression 1,2,3,4,5,6,7. The recent discovery of thousands of lncRNAs in association with specific chromatin modification complexes, such as Polycomb Repressive Complex 2 (PRC2) that mediates histone H3 lysine 27 trimethylation (H3K27me3), suggests broad roles for numerous lncRNAs in managing chromatin states in a gene-specific fashion 8,9. While some lncRNAs are thought to work in cis on neighboring genes, other lncRNAs work in trans to regulate distantly located genes. For instance, Drosophila lncRNAs roX1 and roX2 bind numerous regions on the X chromosome of male cells, and are critical for dosage compensation 10,11. However, the exact locations of their binding sites are not known at high resolution. Similarly, human lncRNA HOTAIR can affect PRC2 occupancy on hundreds of genes genome-wide 3,12,13, but how specificity is achieved is unclear. LncRNAs can also serve as modular scaffolds to recruit the assembly of multiple protein complexes. The classic trans-acting RNA scaffold is the TERC RNA that serves as the template and scaffold for the telomerase complex 14; HOTAIR can also serve as a scaffold for PRC2 and a H3K4 demethylase complex 13.
Prior studies mapping RNA occupancy at chromatin have revealed substantial insights 15,16, but only at a single gene locus at a time. The occupancy sites of most lncRNAs are not known, and the roles of lncRNAs in chromatin regulation have been mostly inferred from the indirect effects of lncRNA perturbation. Just as chromatin immunoprecipitation followed by microarray or deep sequencing (ChIP-chip or ChIP-seq, respectively) has greatly improved our understanding of protein-DNA interactions on a genomic scale, here we illustrate a recently published strategy to map long RNA occupancy genome-wide at high resolution 17. This method, Chromatin Isolation by RNA Purification (ChIRP) (Figure 1), is based on affinity capture of target lncRNA:chromatin complex by tiling antisense-oligos, which then generates a map of genomic binding sites at a resolution of several hundred bases with high sensitivity and low background. ChIRP is applicable to many lncRNAs because the design of affinity-probes is straightforward given the RNA sequence and requires no knowledge of the RNA's structure or functional domains.

Chromatin Isolation by RNA Purification ChIRP

ChIRP is a novel and rapid technique to map genomic binding sites of long noncoding RNAs (lncRNAs). The method takes advantage of the specificity of anti-sense tiling oligonucleotides to allow the enumeration of lncRNA-bound genomic sites.

RNA精製によってクロマチンの単離（チャープ）

Long noncoding RNAs are key regulators of chromatin states for important biological processes such as dosage compensation, imprinting, and developmental ...

Substrate Generation for Endonucleases of CRISPR/Cas Systems

The interaction of viruses and their prokaryotic hosts shaped the evolution of bacterial and archaeal life. Prokaryotes developed several strategies to evade viral attacks that include restriction modification, abortive infection and CRISPR/Cas systems. These adaptive immune systems found in many Bacteria and most Archaea consist of clustered regularly interspaced short palindromic repeat (CRISPR) sequences and a number of CRISPR associated (Cas) genes (Fig. 1) 1-3. Different sets of Cas proteins and repeats define at least three major divergent types of CRISPR/Cas systems 4. The universal proteins Cas1 and Cas2 are proposed to be involved in the uptake of viral DNA that will generate a new spacer element between two repeats at the 5' terminus of an extending CRISPR cluster 5. The entire cluster is transcribed into a precursor-crRNA containing all spacer and repeat sequences and is subsequently processed by an enzyme of the diverse Cas6 family into smaller crRNAs 6-8. These crRNAs consist of the spacer sequence flanked by a 5' terminal (8 nucleotides) and a 3' terminal tag derived from the repeat sequence 9. A repeated infection of the virus can now be blocked as the new crRNA will be directed by a Cas protein complex (Cascade) to the viral DNA and identify it as such via base complementarity10. Finally, for CRISPR/Cas type 1 systems, the nuclease Cas3 will destroy the detected invader DNA 11,12 .
These processes define CRISPR/Cas as an adaptive immune system of prokaryotes and opened a fascinating research field for the study of the involved Cas proteins. The function of many Cas proteins is still elusive and the causes for the apparent diversity of the CRISPR/Cas systems remain to be illuminated. Potential activities of most Cas proteins were predicted via detailed computational analyses. A major fraction of Cas proteins are either shown or proposed to function as endonucleases 4.
Here, we present methods to generate crRNAs and precursor-cRNAs for the study of Cas endoribonucleases. Different endonuclease assays require either short repeat sequences that can directly be synthesized as RNA oligonucleotides or longer crRNA and pre-crRNA sequences that are generated via in vitro T7 RNA polymerase run-off transcription. This methodology allows the incorporation of radioactive nucleotides for the generation of internally labeled endonuclease substrates and the creation of synthetic or mutant crRNAs. Cas6 endonuclease activity is utilized to mature pre-crRNAs into crRNAs with 5'-hydroxyl and a 2',3'-cyclic phosphate termini.

CRISPR/Cas systems mediate adaptive immunity in Bacteria and Archaea. Many Cas proteins are proposed to act as endoribonucleases acting on crRNA precursors of varying length. Here we illustrate three different approaches to generate pre-crRNA substrates for the biochemical analysis of Cas endonuclease activity.

CRISPR / CASシステムの酵素のための基質の生成

The  interaction of viruses and their prokaryotic hosts shaped the evolution of  bacterial and archaeal life. Prokaryotes developed several strategies to ...

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems including plants, invertebrates and vertebrates. There are various methods to achieve RNAi in vivo4,5. For example, the target gene may be transformed into an RNAi vector, and then either permanently or transiently transformed into cell lines or primary cells to achieve gene knockdown effects; alternatively synthesized double-strand oligonucleotides from specific target genes (RNAi oligos) may be transiently transformed into cell lines or primary cells to silence target genes; or synthesized double-strand RNA molecules may be microinjected into an organism. Since the nematode C. elegans uses bacteria as a food source, feeding the animals with bacteria expressing double-strand RNA against target genes provides a viable strategy6. Here we present an RNAi feeding method to score body size phenotype. Body size in C. elegans is regulated primarily by the TGF- &beta; - like ligand DBL-1, so this assay is appropriate for identification of TGF-&beta; signaling components7. We used different strains including two RNAi hypersensitive strains to repeat the RNAi feeding experiments. Our results showed that rrf-3 strain gave us the best expected RNAi phenotype. The method is easy to perform, reproducible, and easily quantified. Furthermore, our protocol minimizes the use of specialized equipment, so it is suitable for smaller laboratories or those at predominantly undergraduate institutions.

Using RNA-mediated Interference Feeding Strategy to Screen for Genes Involved in Body Size Regulation in the Nematode C. elegans

We demonstrate how to use the RNAi feeding technique to knock down target genes and score body size phenotype in C. elegans. This method could be used for a large scale screen to identify potential genetic components of interest, such as those involved in body size regulation by DBL-1/TGF-&beta; signaling.

線虫における身体サイズ調節に関与する遺伝子をスクリーニングするための戦略を給餌RNA媒介干渉を用いた<em&gt; Cエレガンス</em

Double-strand RNA-mediated interference (RNAi) is an effective strategy to knock down target gene expression1-3. It has been applied to many model systems ...

Depletion of Ribosomal RNA for Mosquito Gut Metagenomic RNA-seq

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity and functionality of the gut community will provide insight into the effects of gut microbiota on mosquito life traits. Metagenomic RNA-Seq has become an important tool to analyze transcriptomes from various microbes present in a microbial community. Messenger RNA usually comprises only 1-3% of total RNA, while rRNA constitutes approximately 90%. It is challenging to enrich messenger RNA from a metagenomic microbial RNA sample because most prokaryotic mRNA species lack stable poly(A) tails. This prevents oligo d(T) mediated mRNA isolation. Here, we describe a protocol that employs sample derived rRNA capture probes to remove rRNA from a metagenomic total RNA sample. To begin, both mosquito and microbial small and large subunit rRNA fragments are amplified from a metagenomic community DNA sample. Then, the community specific biotinylated antisense ribosomal RNA probes are synthesized in vitro using T7 RNA polymerase. The biotinylated rRNA probes are hybridized to the total RNA. The hybrids are captured by streptavidin-coated beads and removed from the total RNA. This subtraction-based protocol efficiently removes both mosquito and microbial rRNA from the total RNA sample. The mRNA enriched sample is further processed for RNA amplification and RNA-Seq.

A ribosomal RNA (rRNA) depletion protocol was developed to enrich messenger RNA (mRNA) for RNA-seq of the mosquito gut metatranscriptome. Sample specific rRNA probes, which were used to remove rRNA via subtraction, were created from the mosquito and its gut microbes. Performance of the protocol can result in the removal of approximately 90-99% of rRNA.

モスキートガットメタゲノムRNA-seqのためのリボソームRNAの枯渇

The mosquito gut accommodates dynamic microbial communities across different stages of the insect's life cycle. Characterization of the genetic capacity ...

RNA Catalyst as a Reporter for Screening Drugs against RNA Editing in Trypanosomes

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been made in identifying the components of the editosome complex that catalyze RNA editing. However, it is still not clear how those proteins work together. Chemical compounds obtained from a high-throughput screen against the editosome may block or affect one or more steps in the editing cycle. Therefore, the identification of new chemical compounds will generate valuable molecular probes for dissecting the editosome function and assembly. In previous studies, in vitro editing assays were carried out using radio-labeled RNA. These assays are time consuming, inefficient and unsuitable for high-throughput purposes. Here, a homogenous fluorescence-based &#8220;mix and measure&#8221; hammerhead ribozyme in vitro reporter assay to monitor RNA editing, is presented. Only as a consequence of RNA editing of the hammerhead ribozyme a fluorescence resonance energy transfer (FRET) oligoribonucleotide substrate undergoes cleavage. This in turn results in separation of the fluorophore from the quencher thereby producing a signal. In contrast, when the editosome function is inhibited, the fluorescence signal will be quenched. This is a highly sensitive and simple assay that should be generally applicable to monitor in vitro RNA editing or high throughput screening of chemicals that can inhibit the editosome function.

A highly sensitive ribozyme-based assay, applicable to high-throughput screening of chemicals targeting the unique process of RNA editing in trypanosomatid pathogens, is described in this paper. Inhibitors can be used as tools for hypothesis-driven analysis of the RNA editing process and ultimately as therapeutics.

トリパノソーマにおけるRNA編集に対する薬剤をスクリーニングするためのレポーターとして、RNA触媒

Substantial progress has been made in determining the mechanism of mitochondrial RNA editing in trypanosomes. Similarly, considerable progress has been ...

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully annotated genome sequence, transposon-based transgenesis, and effective RNA interference (RNAi). Among these advantages, RNAi-based gene knockdown techniques are at the core of Tribolium research. T. castaneum show a robust systemic RNAi response, making it possible to perform RNAi at any life stage by simply injecting double-stranded RNA (dsRNA) into the beetle&#8217;s body cavity.
In this report, we provide an overview of our larval RNAi technique in T. castaneum. The protocol includes (i) isolation of the proper stage of T. castaneum larvae for injection, (ii) preparation for the injection setting, and (iii) dsRNA injection. Larval RNAi is a simple, but powerful technique that provides us with quick access to loss-of-function phenotypes, including multiple gene knockdown phenotypes as well as a series of hypomorphic phenotypes. Since virtually all T. castaneum tissues are susceptible to extracellular dsRNA, the larval RNAi technique allows researchers to study a wide variety of tissues in diverse contexts, including the genetic basis of organismal responses to the outside environment. In addition, the simplicity of this technique stimulates more student involvement in research, making T. castaneum an ideal genetic system for use in a classroom setting.

Larval RNA Interference in the Red Flour Beetle, Tribolium castaneum

RNA interference (RNAi)-based gene knockdown techniques are at the core of Tribolium research. Here, we provide an overview of our larval RNAi technique in Tribolium castaneum. Larval RNAi is a simple, but powerful technique that provides quick access to loss-of-function phenotypes, allowing researchers to study gene functions in diverse contexts.

レッドフラワービートル幼虫RNA干渉、<em&gt;コクヌストモドキ</em

The red flour beetle, Tribolium&#160;castaneum, offers a repertoire of experimental tools for genetic and developmental studies, including a fully ...

Production of Double-stranded DNA Ministrings

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ministrings possess a heightened safety profile and also efficiently deliver DNA cargo to targeted cells. Conventional DNA vectors carry undesirable prokaryotic sequences, including antibiotic resistance genes, CpG motifs, and bacterial origins of replication, which may lead to the stimulation of host immunological responses. The bioavailability of conventional DNA vectors is also compromised due to their larger molecular size. Their circular nature may also impart chromosomal integration, leading to insertional mutagenesis.
Bacterial sequences are excised from DNA minivectors, leaving only the gene of interest (GOI) and necessary eukaryotic expression elements. Our LCC DNA minivectors, or DNA ministrings, are devoid of immunogenic bacterial sequences; therefore improving their bioavailability and GOI expression. In the event of vector integration into the chromosome, the LCC DNA ministring will lethally disrupt the host chromosome, thereby removing the potentially dangerous mutant from the proliferating cell population. Consequently, DNA ministrings offer the benefits of &#39;minicircle&#39; DNA while eliminating the potential for undesirable vector integration events. In comparison to conventional plasmids and their isogenic circular covalently closed (CCC) counterparts, DNA ministrings demonstrate superior bioavailability, transfection efficiency, and cytoplasmic kinetics - they thus require lower amounts of cationic surfactants for effective transfection of target cells.
We have constructed a one-step inducible in vivo system for the production of DNA ministrings in Escherichia coli that is simple to use, rapid, and scalable.

The linear covalently closed (LCC) DNA minivector (DNA ministring) is a non-viral gene delivery vector offering high transfection efficiency and is relevant to any DNA delivery application. The production system is simple, rapid, and scalable in vivo. The following protocol provides visual step-by-step instructions for the production of DNA ministrings.

二本鎖DNA Ministringsの生産

We constructed linear covalently closed (LCC) DNA minivectors as a non-viral gene-delivery vector alternative produced via a simple platform in vivo. DNA ...

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression regulation at posttranscriptional levels by affecting the stability and translational efficiency of target mRNAs. RNA pull-down technique has been widely used to study RNA-protein interaction, which is necessary to elucidate the mechanism underlying RBPs&#39; as well as long non-coding RNAs&#39; (lncRNAs) function. However, the high lipid abundance in adipocytes poses a technical challenge in conducting this experiment. Here a detailed RNA pull-down protocol is optimized for primary adipocyte culture. An RNA fragment from androgen receptor&#39;s (AR) 3&#39; untranslated region (3&#39;UTR) containing an adenylate-uridylate-rich elementwas used as an example to demonstrate how to retrieve its RBP partner, HuR protein, from adipocyte lystate. The method described here can be applied to detect the interactions between RBPs and noncoding RNAs, as well as between RBPs and coding RNAs.

Detection of RNA-binding Proteins by In Vitro RNA Pull-down in Adipocyte Culture

An RNA pull-down protocol is optimized here for detection of interactions between RNA-binding proteins (RBPs) and noncoding as well as coding RNAs. An RNA fragment from androgen receptor (AR) was used as an example to demonstrate how to retrieve its RBP from lystate of primary brown adipocytes.

RNA結合タンパク質の検出によって、<em&gt;インビトロ</em脂肪細胞培養における&gt; RNAプルダウン

RNA-binding proteins (RBPs) are emerging as a regulatory layer in the development and function of adipose. RBPs play a key role in the gene expression ...

TChIP-Seq: Cell-Type-Specific Epigenome Profiling

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological functions have been extensively studied. Indeed, the advent of next-generation deep sequencing and chromatin immunoprecipitation (ChIP) via specific histone modification antibodies has revolutionized our view of epigenetic regulation across the genome. Conversely, tissues typically consist of diverse cell types, and their complex mixture poses analytic challenges to investigating the epigenome in a particular cell type. To address the cell type-specific chromatin state in a genome-wide manner, we recently developed tandem chromatin immunoprecipitation sequencing (tChIP-Seq), which is based on the selective purification of chromatin by tagged core histone proteins from cell types of interest, followed by ChIP-Seq. The goal of this protocol is the introduction of best practices of tChIP-Seq. This technique provides a versatile tool for tissue-specific epigenome investigation in diverse histone modifications and model organisms.

We describe a step-by-step protocol for tandem chromatin immunoprecipitation sequencing (tChIP-Seq) that enables the analysis of cell-type-specific genome-wide histone modification.

TChIP-Seq: 細胞型特異エピゲノムのプロファイリング

Epigenetic regulation plays central roles in gene expression. Since histone modification was discovered in the 1960s, its physiological and pathological ...

RNA 干渉法の活用: ゴキブリのリポソームキャリアで二本鎖 RNA の経口デリバリー

要約

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この動画の章

HeLa細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

RNA精製によってクロマチンの単離（チャープ）

CRISPR / CASシステムの酵素のための基質の生成

線虫における身体サイズ調節に関与する遺伝子をスクリーニングするための戦略を給餌RNA媒介干渉を用いた

モスキートガットメタゲノムRNA-seqのためのリボソームRNAの枯渇

トリパノソーマにおけるRNA編集に対する薬剤をスクリーニングするためのレポーターとして、RNA触媒

レッドフラワービートル幼虫RNA干渉、

二本鎖DNA Ministringsの生産

RNA結合タンパク質の検出によって、

TChIP-Seq: 細胞型特異エピゲノムのプロファイリング

RNA 干渉法の活用: ゴキブリのリポソーム キャリアで二本鎖 RNA の経口デリバリー

要約

さらに動画を探す

この動画の章

HeLa細胞のトランスフェクションに続いてGaussiaルシフェラーゼmRNA のインビトロ転写とキャッピングの

RNA精製によってクロマチンの単離（チャープ）

CRISPR / CASシステムの酵素のための基質の生成

線虫における身体サイズ調節に関与する遺伝子をスクリーニングするための戦略を給餌RNA媒介干渉を用いた

モスキートガットメタゲノムRNA-seqのためのリボソームRNAの枯渇

トリパノソーマにおけるRNA編集に対する薬剤をスクリーニングするためのレポーターとして、RNA触媒

レッドフラワービートル幼虫RNA干渉、

二本鎖DNA Ministringsの生産

RNA結合タンパク質の検出によって、

TChIP-Seq: 細胞型特異エピゲノムのプロファイリング

RNA 干渉法の活用: ゴキブリのリポソームキャリアで二本鎖 RNA の経口デリバリー