C.Y.W. received support from the National Health Research Institute (NHRI-EX101-9925SC), the National Science Council (101-2314-B-182-100-MY3, 101-2314-B-182A-009), and Chang Gung Memorial Hospital (CMRPG3B1643, CMRPG3D1002, CMRPG3D0581, CMRPG380091, and CMRPG3C1763).

注：6レベルのrRNA、mRNAの、および他の小RNAを含むトータルRNAメートル。リボソームRNAが豊富メートル6 Aの変更を持っているので、メートルの測定6 Aレベルは、この事実を考慮する必要があります。 1. RNAの単離<ol><li> （ペーパータオル上に噴霧）リボヌクレアーゼ除染液を使用して、ピペットと実験台の表面を拭いてください。ヌクレアーゼフリー水で濡れた紙タオル、再びピペットおよび実験台の表面を拭いてください。 </li><li> RNA抽出<ol><li>全RNA単離<ol><li>オーバーヘッド撹拌機を用いて、4℃に維持し、RNA単離溶液1mLに組織の50~100ミリグラムまたは5~10×10 6個の細胞をホモジナイズします。 注：その他の組織（100-150 mg）をため、その中に低いRNA濃度のマウスからの脂肪組織サンプルのために必要となる場合があります。サンプルは、マウスの心臓、肝臓、骨格筋、肺、脳から供給、およびマクロファージは、以前4採用されています。 </li><li>に室温で5分間、試料ホモジネートをcubate。 </li><li>サンプルホモジネートの1ミリリットル当たり1-ブロモ-3-クロロプロパン（BCP）の100μLを加えます。 15秒間激しくチューブを振ると、製造元の指示24に従って全RNAを単離するために進んでください。 </li></ol></li><li>単離された全RNAからポリアデニル化mRNA精製<ol><li>入手可能なキットまたはプロトコルを使用してポリアデニル化mRNAを精製します。 <ol><li>次の各溶液を再懸濁するために徹底的に振る：結合液、オリゴ（dT）ポリスチレンビーズ、および洗浄溶液を2倍。オリゴ（dT）は使用前に室温に戻しビーズことを確認してください。 </li><li>加熱ブロック中で70℃にチューブや熱に準備あたりの溶出溶液の120μLを転送します。 </li><li>マイクロ遠心チューブに全RNAの500μgのまでピペット。ヌクレアーゼフリー水で、250μLに音量を調整します。 </li><li>全RNAを2倍結合溶液の250μLを追加しますので、内容物を混合するための簡単リューションと渦。 </li><li>オリゴの15μLを加える（DT）ビーズと渦が完全に混合します。 </li><li> 3分間70℃で混合物をインキュベートします。 </li><li>加熱ブロックからサンプルを取り出して、それを10分間室温で放置しました。 </li><li> 2分間15,000×gで遠心分離します。 </li><li>慎重に約50μLを残す、上清を除去します。 </li><li>ピペッティングによりペレットを再懸濁するために洗浄溶液の500μLを追加します。 </li><li>スピンフィルター/コレクションチューブセットにサスペンションをピペット。 </li><li> 2分間15,000×gで遠心分離します。コレクションチューブから列を削除し、フロースルーを捨てます。コレクションチューブにカラムを返します。 </li><li>スピンフィルター上に洗浄溶液500μLをピペット。 </li><li> 15,000 xでの遠心分離 2分間グラム。 </li><li>新鮮なコレクションチューブにスピンフィルターを転送します。 </li><li>加熱された溶出液のピペット50μL70°Cにスピンフィルタの中心へ。 </li><li> 70℃で2〜5分間インキュベートします。 1分間15,000×gで遠心分離します。 </li><li>スピンフィルターの中央に70℃に加熱し、溶出液の追加の50μLをピペット。 </li><li>繰り返しステップ1.2.2.1.18。 </li></ol></li><li> 100μg/ mLのグリコーゲン、3 M酢酸ナトリウム緩衝液（pH 5.2）、および無水エタノールの2.5倍量の0.1ボリュームを追加します。 -80℃で一晩沈殿させます。 </li><li> 4℃で25分間、15,000×gで遠心分離します。上清を捨てます。 </li><li> 75％エタノール1mLでペレットを洗浄。 4℃で15分間、15,000×gで遠心分離します。慎重にエタノールを除去。 </li><li> 3-5分間空気中で乾燥しました。 </li><li>ペレットを溶解するためにヌクレアーゼフリー水10μLを追加します。 </li><li> -70℃で保管してください。 </li></ol></li></ol></li><li> RNA資格および定量<ol><li>分光光度計を使用して、NotIでによってRNA濃度を測定260 nmおよび280 nmの吸光度をngの。 A 260 / A 280比は1.8〜2.2でなければなりません。 </li><li> 0.8％アガロースゲル電気泳動25を用いて、RNAサンプルの品質を確認してください。 注：無傷の真核生物の全RNAは、強烈な28Sおよび18S rRNAバンドを示すべきです。良いRNA調製のための18S rRNAのバンド強度対28Sの割合は約2です。 </li></ol></li></ol> 2.ゲル電気泳動および転送注意：バッファ調製プロトコルを表1に示します。 <ol><li>ホルムアルデヒドゲル調製（1％） <ol><li>ジエチル（DEPC）水と、すべての電気機器をすすぎます。 </li><li> DEPC水215 mL中に完全に電子レンジでアガロース2.5gのメルト。 </li><li> 10倍3-（N-モルホリノ）プロパンスルホン酸（MOPS）の12.5 mLの緩衝液および37％ホルムアルデヒドの22.5 mLを加え。 </li><li> 1.5ミリメートルの厚さのくしと電気泳動装置にそれを注ぎます。 </li><li>トン気泡を排除またはプッシュきれいな櫛でのゲルのエッジに裾。 </li><li>ゲルは室温で固化することを可能にします。 </li></ol></li><li>試料調製<ol><li>サンプルバッファの全RNAの1-10μgの11.3μLを混ぜます。 </li><li>サンプルバッファの11.3μLとRNAマーカー（を1μg/μL）の2μgのを混ぜます。 </li><li> 2.2.1および2.2.2からの16μLの総容量にサンプルにヌクレアーゼフリー水を追加します。 </li><li> 5分間60℃で加熱し、次いで氷上で冷やします。 </li><li>氷の上では0.1μg/μLのエチジウムブロマイドが含まれている追跡用色素の4μL、と2.2.3からの試料16μLを混ぜます。 注意：吸入するとエチジウムブロマイドはおそらく催奇形性や毒性があります。エチジウムブロマイド安全データシートをお読みください。 </li></ol></li><li>電気泳動<ol><li> 10×MOPSの200 mLの1×MOPSランニングバッファーを作るためのddH 2 Oの1800 mLにバッファを追加します。 </li><li>ゲルのウェルを洗浄するために、1×MOPSランニングバッファーを使用してください。 </li><li>プレラントン彼は5分間、20Vでゲル。 </li><li>ゲルのウェルにRNAサンプルをロードします。 </li><li>光暴露を避けるためにホイルで覆います。約17時間（一晩）35 Vで実行します</li><li>調べ、UV光下でゲルを撮影。 </li></ol></li><li>移転<ol><li>未使用部分を除去するためにゲルをカットします。 </li><li> 10×SSC緩衝液（20×SSC緩衝液の250ミリリットルとDEPC水の250 mL）を500 mLのを準備します。 </li><li> 50 rpmで振とうしながら20分間、10×SSC緩衝液で2回ゲルを洗浄してください。 </li><li>搬送用トレイ（ 図1）から転送バッファを吸収芯紙、として機能するのに十分な大き1フィルタ紙をカットします。 </li><li>ゲルと同じ大きさにろ紙の4枚をカット。 </li><li>ゲルと同じ大きさに正に帯電したナイロン膜の1シートをカット。 </li><li>ゲル上のノッチと適切な向きを確保するためのマーカーとしての膜をマークします。 </li><li> 15分間、2×SSC緩衝液中で膜を浸します。 </li><li> 500 mLのOを注ぎますF 10倍SSC転送トレイにバッファー。 </li><li>ガラス板を横切って大きなフィルタ紙を置き、転写緩衝液で濾紙を濡らします。 </li><li>ピペットで気泡をロールアウト。 </li><li>転送バッファにプレカットろ紙の2枚を浸し、ステップ2.4.5からろ紙の中央に配置します。任意の気泡を除去。 </li><li>ろ紙上にゲルのトップ側を下にして置きます。 </li><li>ゲルの上に膜を置きます。ゲルとメンブレンのノッチを合わせます。 </li><li>膜の上にプレカット濾紙の2枚を配置し、転写緩衝液で濾紙を濡らします。 </li><li>層の間の任意の気泡を除去。 </li><li>タオルだけゲルと膜を通過バ​​ッファを吸い取ることを保証するために、ゲルの周りにラップを適用します。 </li><li>ろ紙の上にペーパータオルをスタック。 </li><li>タオルの上に特大のガラス板を置きます。 </li><li>スタックの一番上に重量を配置します。 </li><li>RNAは、膜にゲルから転送できるようにするために一晩保管してください。 </li></ol></li></ol> N 6 -methyladenosineの3検出<ol><li>膜架橋<ol><li> 2×SSC緩衝液で湿った膜を保管してください。 </li><li> UVクロスリンカーに10×SSC緩衝液で浸した濾紙の2枚を置きます。 </li><li> RNA吸着側を上に向けて、ろ紙の上に膜を配置します。 </li><li> 「autocrosslinkモード」（12万μJ）を選択し、照射を開始するために、「スタート」ボタンを押してください。 </li><li>ゲルからメンブレンへのRNA転写を確認するために、UVゲルイメージキャッチャーを有する膜およびゲルを調べます。 </li></ol></li><li>免疫ブロット法<ol><li> 1時間室温でトゥイーン20（TBST）緩衝液を用いてトリス緩衝生理食塩水、5％脱脂乳で膜をブロックします。 </li><li> 15分間、TBST緩衝液で3回洗浄します。 </li><li>一晩メートル6 A（中膜をインキュベート&lt;EM&gt; N 6 -methyladenosine）抗体溶液（1：5％脱脂乳TBSTバッファー1,000）4℃。 </li><li> 15分間、TBST緩衝液で3回膜を洗浄します。 </li><li>室温で1時間（5％の脱脂乳TBST緩衝液中2,000 1）HRP結合ロバ抗ウサギ抗体溶液中で膜をインキュベートします。 </li><li> 15分間、TBST緩衝液で3回膜を洗浄します。 </li><li>強化された化学発光基質（膜の1cm 2当たり0.125ミリリットル）を適用します。 </li><li>製造元の指示26に従って、デジタルイメージャの最適な設定で化学発光をキャプチャします。 </li></ol></li><li>メートル6定量<ol><li> ImageJソフトウェアを使用して相対メートル6 A化学発光強度を測定します。 <ol><li> ImageJソフトウェアのメニューで、該当するファイルを開くには、「ファイル」オプションを選択します。 </li><li> ImageJのから「矩形」ツールを選択し、周囲に枠を描きます信号。 </li><li>リボソームRNAは、実験の焦点でない場合は、計算のための18Sおよび28SリボソームRNAメートル6 Aバンドを避けます。 </li><li>選択された車線を確認するために「コマンド」と「1」をクリックしてください。 </li><li>押して「コマンド」と「3」が選択されたプロットを表示します。 </li><li> 「ストレート」ツールをクリックし、セグメント化された領域を分離するために線を引きます。 </li><li>測定値を記録するために「ワンド」ツールをクリックします。 </li><li>データをエクスポートします。 </li><li>臭化エチジウム染色から18SリボソームRNAのバンドを持つメートル6 Aのレベルを標準化します。 </li></ol></li></ol></li></ol>

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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=Method+for+site-specific+detection+of+m6A+nucleoside+presence+in+RNA+based+on+high-resolution+melting+(HRM)+analysis.">Method for site-specific detection of m6A nucleoside presence in RNA based on high-resolution melting (HRM) analysis.</a> Nucleic Acids Res. 42 (4), 27 (2014).</li><li>Dominissini, D., 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=Topology+of+the+human+and+mouse+m6A+RNA+methylomes+revealed+by+m6A-seq.">Topology of the human and mouse m6A RNA methylomes revealed by m6A-seq.</a> Nature. 485 (7397), 201-206 (2012).</li><li>Linder, B., 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=Single-nucleotide-resolution+mapping+of+m6A+and+m6Am+throughout+the+transcriptome.">Single-nucleotide-resolution mapping of m6A and m6Am throughout the transcriptome.</a> Nat Methods. 12 (8), 767-772 (2015).</li><li>Fu, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+Quantification+of+Methylated+Cytidine+and+Adenosine+in+Cellular+and+Tissue+RNA+by+Nano-Flow+Liquid+Chromatography-Tandem+Mass+Spectrometry+Coupled+with+the+Stable+Isotope-Dilution+Method.">Simultaneous Quantification of Methylated Cytidine and Adenosine in Cellular and Tissue RNA by Nano-Flow Liquid Chromatography-Tandem Mass Spectrometry Coupled with the Stable Isotope-Dilution Method.</a> Anal Chem. 87 (15), 7653-7659 (2015).</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=Nondenaturing+agarose+gel+electrophoresis+of+RNA.">Nondenaturing agarose gel electrophoresis of RNA.</a> Cold Spring Harb Protoc. 2010 (6), 5445 (2010).</li><li>Wang, C. 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著者らは、開示することは何もありません。

Modifications of RNA have important roles in cellular function and physiology. The current understanding of the regulation, function, and homeostasis of these modifications is still being explored and expanded8. Therefore, a precise and gold-standard method to evaluate the modifications of RNA is needed. The modified northern blotting method provides precise quantification of RNA modifications and clear delineation of the modifications in diverse RNAs. Although the method requires at least 3 days, it can be standardized and can be used in various experimental designs. Moreover, with different antibodies, it can detect different RNA modifications27.
It is important to separate different RNAs when analyzing RNA modifications. Ribosomal RNA comprises a large portion of the total amount of RNA28,29. The results from analyzing RNA modifications only in total RNA will represent mostly the changes of rRNA. Methylation and other such modifications of rRNA could potentially mask the changes in other RNAs. With the procedure of gel separation, the modifications of mRNA and other small RNAs can be more accurately analyzed.
Transcriptome-wide mapping with m6A immunoprecipitation and sequencing provides detailed insight into the modification of each type of RNA22. It provides information on the specific RNAs and a resolution of around 80-120 bp. Although m6A-Seq can compare the modifications between different experimental conditions, the selection of proper standards and controls for such experiments is difficult18. Immunoprecipitation is difficult to reproduce, often giving significant variations amongst repeats. Moreover, m6A-Seq requires the fragmentation of RNA samples before immunoprecipitation and sequencing30. The fragmentation process could potentially induce undue influences on the original RNA modifications. If the experiment does not need the specific gene's information but requires different conditions for comparison, the current method provides better visualization and control for diverse experimental setups. 
Modification of RNA is an important step in regulating transcriptional control31. However, the homeostasis and the regulatory mechanisms of various RNA modifications under diverse physiological realms are still unclear. Using the present modified northern blotting method, different RNA modifications can be quantified and compared. Furthermore, the changes and regulations of RNA modifications can be investigated in greater detail. In the future, it could also be possible to combine the experimental data from both the classical northern blotting and the modified northern blotting protocols, providing greater insights into RNA biology.
The most important factor determining the success of the modified northern blotting protocol is the integrity of the RNA sample. RNAs with some amount of degradation may yield good classical northern blotting results, but this could potentially have significant impact on the modified northern blotting results. The modifications of RNA in different tissues or cell lines could also vary significantly. It is important to test the suitable RNA sample loads for different tissues before performing the final experiments.
As blotting procedures have been traditionally named after Dr. Southern and different geographical directions, we propose the name "northeastern" blotting for the current technique.

Dynamic and reversible RNA modifications have important roles in RNA homeostasis. Four decades ago, N6-methyladenosine (m6A) modifications were found to be abundant in eukaryotic transcriptomes1. They have diverse functions in messenger RNA (mRNA), ribosomal RNA (rRNA), small nucleolar RNA, transfer RNA, and microRNA2. The m6A modifications of mRNA influence their splicing3, translation4, degradation5, and localization2. Moreover, they affect ribosome biogenesis and microRNA function6. The evolutionary conservation of m6A modifications of RNA is noted in unicellular bacteria to multi-cellular humans7. Delineation of the roles of m6A modifications is currently under extensive exploration. The efforts are expected to provide new insights into transcription control. Recent studies reveal that other chemical modifications of mRNA8 also play critical roles in RNA metabolism. 
Circadian rhythms9, stem cell pluripotency10, triglyceride metabolism4, fibrosis11, obesity12, and major depression13 are a few examples of processes where m6A modifications are known to control outcomes. Many circadian clock gene transcripts have m6A sites14. Modulation of m6A methylase or demethylase elicits circadian period changes15. Mettl3, an m6A transferase, is a regulator for stem cell pluripotency. A deficiency of Mettl3 leads to early embryonic lethality and aberrant lineage priming at the post-implantation stage10. A deficiency of fat mass- and obesity-associated (FTO), an m6A demethylase, in adipocytes affects fatty acid mobilization and body weight through posttranscriptional regulation of Angptl44. These studies reveal that m6A not only controls mRNA processing, but also plays critical roles in embryological development and patho-physiology. The function of m6A modifications holds implications for therapeutic considerations in the future.
Several methods are available to measure m6A modifications of RNA16-18. Traditionally, thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC) are used to study the distribution of m6A in several RNAs19,20. Mass spectrometry is a sensitive tool for the detection of m6A modifications in RNA. However, the RNA needs to be excised by RNase into short fragments before analysis by mass spectrometry21. Methyl-RNA immunoprecipitation and sequencing (m6A-Seq)22 immunoprecipitates fragmented RNAs with m6A-specific antibodies and performs parallel RNA sequencing. This method generates transcriptome-wide m6A landscapes. High-resolution mapping of m6A individual-nucleotide-resolution cross-linking and immunoprecipitation (miCLIP) further maps m6A modifications at a single-nucleotide resolution23. Both methods provide details of m6A modification across the whole transcriptome, with specific genes' information. However, quantifications and standardization in both methods are difficult if experiments require the comparison of multiple conditions. Moreover, fragmentations of RNA for m6A-Seq alter the original RNA structure, which may affect native m6A levels. To detect global m6A modifications of RNA and their changes under different experimental conditions, we report a method that employs a modified northern blotting protocol. This method resolves RNA by molecular weight, using gel electrophoresis18. This procedure provides better standardization and quantifications for experiments that involve multiple conditions or samples. It also provides specific m6A modification information for different RNAs, whether rRNA, mRNA, or microRNA.

<table border="0" cellpadding="0" cellspacing="0" width="598">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNaseZap solution</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM9782</td>
			<td style="width:167px;">Protocol 1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">TRI Reagent solution</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM9738</td>
			<td>Protocol 1.2.1.1</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">1-Bromo-3-chloropropane (BCP)</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">B9673</td>
			<td>Protocol 1.2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol</td>
			<td style="width:96px;">JTbaker</td>
			<td style="width:119px;">8006</td>
			<td>Protocol 1.2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Isopropanol</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">I9516</td>
			<td>Protocol 1.2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GenElute mRNA Miniprep Kit</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">MRN70</td>
			<td>Protocol 1.2.2.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Glycogen</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM9510</td>
			<td>Protocol 1.2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium acetate</td>
			<td style="width:96px;">Fluka</td>
			<td style="width:119px;">71183</td>
			<td>Protocol 1.2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nuclease-free water</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM9930</td>
			<td>Protocol 1.2.2.6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">DEPC</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">D5758</td>
			<td>Protocol 2.1.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Agarose</td>
			<td style="width:96px;">JT Baker</td>
			<td style="width:119px;">A426</td>
			<td>Protocol 2.1.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MOPS</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">M1254</td>
			<td>Protocol 2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">37% formaldehyde Solution</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">F8775</td>
			<td>Protocol 2.1.3</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">EDTA, Disodium Salt</td>
			<td style="width:96px;">JT Baker</td>
			<td style="width:119px;">8993</td>
			<td>Protocol 2.1.3 10X MOPS buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">formamide</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">F7503</td>
			<td>Protocol 2.2.1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNA Millennium Marker</td>
			<td style="width:96px;">Ambion</td>
			<td style="width:119px;">AM7150</td>
			<td>Protocol 2.2.2</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethidium Bromide</td>
			<td style="width:96px;">Amresco</td>
			<td style="width:119px;">X328</td>
			<td>Protocol 2.2.5</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ficoll 400</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">17-0300-10</td>
			<td>Protocol 2.2.5 tracking dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Bromophenol blue</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">114391</td>
			<td>Protocol 2.2.5 tracking dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Xylene Cyanol</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">X4126</td>
			<td>Protocol 2.2.5 tracking dye</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Sodium chloride</td>
			<td style="width:96px;">JT Baker</td>
			<td style="width:119px;">3624</td>
			<td>Protocol 2.4.2 20X SSC buffer</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trisodium citrate</td>
			<td style="width:96px;">Sigma</td>
			<td style="width:119px;">S1804</td>
			<td>Protocol 2.4.2 20X SSC buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">filter paper</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN6101M</td>
			<td>Protocol 2.4.4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">GE Hybond-N+ membrane</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">RPN303B</td>
			<td>Protocol 2.4.6</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Stratalinker UV Crosslinker 2400</td>
			<td style="width:96px;">Stratagene</td>
			<td style="width:119px;">400075</td>
			<td>Protocol 3.1.2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Gel Catcher 1500</td>
			<td style="width:96px;">ANT Technology</td>
			<td style="width:119px;">Gel Catcher 1500</td>
			<td>Protocol 3.1.5</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anti-m6A (N6-methyladenosine)</td>
			<td style="width:96px;">Synaptic Systems</td>
			<td style="width:119px;">202003</td>
			<td>Protocol 3.2.3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amersham ECL Anti-Rabbit IgG, HRP-linkd whole Ab</td>
			<td style="width:96px;">GE Healthcare</td>
			<td style="width:119px;">NA934</td>
			<td>Protocol 3.2.5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">ChemiDoc MP System</td>
			<td style="width:96px;">BIO-RAD</td>
			<td style="width:119px;">1708280</td>
			<td>Protocol 3.2.8</td>
		</tr>
	</tbody>
</table>

a method for measuring rna n6-methyladenosine modifications in cells and tissues

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies have revealed that these reversible RNA modifications affect RNA splicing, translation, degradation, and localization. Multiple physiological processes, like circadian rhythms, stem cell pluripotency, fibrosis, triglyceride metabolism, and obesity are also controlled by m6A modifications. Immunoprecipitation/sequencing, mass spectrometry, and modified northern blotting are some of the methods commonly employed to measure m6A modifications. Herein, we present a northeastern blotting technique for measuring m6A modifications. The current protocol provides good size separation of RNA, better accommodation and standardization for various experimental designs, and clear delineation of m6A modifications in various sources of RNA. While m6A modifications are known to have a crucial impact on human physiology relating to circadian rhythms and obesity, their roles in other (patho)physiological states are unclear. Therefore, investigations on m6A modifications have immense possibility to provide key insights into molecular physiology.

通常の明暗概日相で14日後に、野生型マウスは、一定の暗闇の中に入れました。肝臓からのRNAは、4時間間隔でサンプリングし、変更されたノーザンブロット法で検討しました。 rRNA、mRNAを、と小さなRNAのメチル化は、明らかに（ 図2）を検出しました。別の概日時間（CT）との間の比較は、正確に、18S rRNAの標準を用いて計算することができます。 rRNAを、mRNAを、および低分子RNAでメートル6 Aレベルの堅牢な概日振動がありました。 リボソームRNAによって差し出さ干渉を回避するために、ポリアデニル化RNAは、ステップ1.2.2のように、精製することができます。精製後、rRNAが大きく、他のRNA（ 図3）のより良好な視覚化を可能にするために除去することができます。 <img alt="図1" src="/files/ftp_upload/54672/54672fig1.jpg" /> <stron<html>グラム&gt;図1：ブロットトランスファーユニットの組み立て。メンブレンへのRNAをブロッティング用のキャピラリートランスファーユニットが示されています。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54672/54672fig1large.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> <img alt="図2" src="/files/ftp_upload/54672/54672fig2.jpg" /> 図2：C57BL / 6Jマウスにおけるメートル6 Aレベルの概日リズム。この図はメートル6野生型マウスの肝臓からの全RNAのブロットは、4時間の間隔で、通常の明暗相の14日後に暗暗期の最初の日に犠牲に示しています。定量化は、M 6 18Sおよび28S rRNAの間の画像の濃度差を用いて行きました。メートル6存在量は一番下にホルムアルデヒドゲルから18S rRNAのバンドに標準化しました。ら= &quot;_空白&quot;&gt;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。 <img alt="図3" src="/files/ftp_upload/54672/54672fig3.jpg" /> 図3：メートル6 Aブロットを持つかのrRNAなし。代表的なmが野生型マウスの肝臓からの全RNAおよびポリアデニル化RNAの6 Aブロットが示されています。 <a href="https://ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/54672/54672fig3large.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> <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 1 </td><td colspan="3"> 10×MOPS緩衝液（pH 7.0、光から保護します） </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> MOPS </td><td> 41.85 </td><td>グラム</td><td> 0.2 M </td></tr><tr><td></td><td colspan="3">酢酸ナトリウム</td><td> 4.1 </td><td>グラム</td><td>0.05 M </td></tr><tr><td></td><td colspan="3"> EDTA、二ナトリウム塩</td><td> 3.7 </td><td>グラム</td><td> 0.01 M </td></tr><tr><td></td><td colspan="3"> DEPC水</td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3">合計</td><td> 1 </td><td> L </td><td></td></tr><tr><td></td><td colspan="3">室温で撹拌</td><td></td><td></td><td></td></tr><tr><td> 2 </td><td colspan="3"> 20×SSC緩衝液（pH7.0） </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3">塩化ナトリウム</td><td> 175.3 </td><td>グラム</td><td> 3 M </td></tr><tr><td></td><td colspan="3">クエン酸三ナトリウム</td><td> 88.3 </td><td>グラム</td><td> 0.3 M </td></tr><tr><td></td><td colspan="3"> DEPC水</td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3">合計</td><td> 1 </td><td> L </td><td></td&gt; </tr><tr><td></td><td colspan="3">室温で攪拌し、その後、オートクレーブ</td><td></td><td></td><td></td></tr><tr><td> 3 </td><td colspan="3">追跡用色素</td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10×MOPS緩衝液</td><td> 500 </td><td> μL </td><td> 1倍</td></tr><tr><td></td><td colspan="3">フィコール400 </td><td> 0.75 </td><td>グラム</td><td> 15％ </td></tr><tr><td></td><td colspan="3">ブロモフェノールブルー</td><td> 0.01 </td><td>グラム</td><td> 0.2％ </td></tr><tr><td></td><td colspan="3">キシレンシアノール</td><td> 0.01 </td><td>グラム</td><td> 0.2％ </td></tr><tr><td></td><td colspan="3"> DEPC水</td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3">合計</td><td> 5 </td><td> mLの</td><td></td></tr><tr><td></td><td colspan="3"> -20°Cで保存</td><td></td><td></td><td></td></tr><tr><td> 4 </td><td colspan="3"> DEPC水</td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 1割合を希釈：DEPCの1,000のddH 2 Oに</td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3">室温で一晩攪拌し、その後、オートクレーブ</td><td></td><td></td><td></td></tr><tr><td> 5 </td><td colspan="3">サンプルバッファー（光から保護） </td><td></td><td></td><td></td></tr><tr><td></td><td colspan="3"> 10×MOPS緩衝液</td><td> 200 </td><td> μL </td><td></td></tr><tr><td></td><td colspan="3"> 37％ホルムアルデヒド</td><td> 270 </td><td> μL </td><td></td></tr><tr><td></td><td colspan="3">ホルムアミド</td><td> 660 </td><td> μL </td><td></td></tr><tr><td></td><td colspan="3"> -20°Cで保存</td><td></td><td></td><td></td></tr></tboDY&gt; </table> 表1：バッファおよびソリューションを提供しています。

Watch this Scientific Journal Video about A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues at JoVE.com

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

N 6 -methyladenosine（メートル6 A）RNAに修飾を測定するための修正されたノーザンブロット法が記載されています。現在の方法は、様々な実験デザインの下で多様なRNAおよびコントロールで変更を検出することができます。

RNAの測定方法<em&gt; N</em<sup&gt; 6</sup細胞や組織における&gt; -methyladenosine変更

N6-Methyladenosine (m6A) modifications of RNA are diverse and ubiquitous amongst eukaryotes. They occur in mRNA, rRNA, tRNA, and microRNA. Recent studies ...

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Nanyang Technological University

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JoVE Journal

Biochemistry

A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

10.8K ビュー.  Chang Gung Memorial Hospital, Chang Gung University College of Medicine.  N 6 -methyladenosine（メートル6 A）RNAに修飾を測定するための修正されたノーザンブロット法が記載されています。現在の方法は、様々な実験デザインの下で多様なRNAおよびコントロールで変更を検出することができます。 

ビデオ: A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues

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.

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 ...

生化学

A Method for Measuring Metabolism in Sorted Subpopulations of Complex Cell Communities Using Stable Isotope Tracing

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. Moreover, even cultures of a single cell type may contain cells in distinct metabolic states, such as resting or cycling cells. Methods for measuring metabolic activities of such subpopulations are valuable tools for understanding cellular metabolism. Complex cell populations are most commonly separated using a cell sorter, and subpopulations isolated by this method can be analyzed by metabolomics methods. However, a problem with this approach is that the cell sorting procedure subjects cells to stresses that may distort their metabolism.
To overcome these issues, we reasoned that the mass isotopomer distributions (MIDs) of metabolites from cells cultured with stable isotope-labeled nutrients are likely to be more stable than absolute metabolite concentrations, because MIDs are formed over longer time scales and should be less affected by short-term exposure to cell sorting conditions. Here, we describe a method based on this principle, combining cell sorting with liquid chromatography-high resolution mass spectrometry (LC-HRMS). The procedure involves analyzing three types of samples: (1) metabolite extracts obtained directly from the complex population; (2) extracts of "mock sorted" cells passed through the cell sorter instrument without gating any specific population; and (3) extracts of the actual sorted populations. The mock sorted cells are compared against direct extraction to verify that MIDs are indeed not altered by the cell sorting procedure itself, prior to analyzing the actual sorted populations. We show example results from HeLa cells sorted according to cell cycle phase, revealing changes in nucleotide metabolism.

This article describes a method for studying cellular metabolism in complex communities of multiple cell types, using a combination of stable isotope tracing, cell sorting to isolate specific cell types, and mass spectrometry.

安定同位体のトレースを使用した複雑な細胞のコミュニティのソートされた亜集団で代謝を測定する方法

Mammalian cell types exhibit specialized metabolism, and there is ample evidence that various co-existing cell types engage in metabolic cooperation. ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

複数のマウス組織からの中間体フィラメントタンパク質の単離による老化に関連する翻訳後修飾の研究

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

質量分析を用いた識別 RNA 結合領域のための試料作製

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

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 ...

Dissipative Microgravimetry to Study the Binding Dynamics of the Phospholipid Binding Protein Annexin A2 to Solid-supported Lipid Bilayers Using a Quartz Resonator

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic properties of the absorbed/immobilized mass on sensor surfaces, allowing the measurements of the interaction of proteins with solid-supported surfaces, such as lipid bilayers, in real-time and with a high sensitivity. Annexins are a highly conserved group of phospholipid-binding proteins that interact reversibly with the negatively charged headgroups via the coordination of calcium ions. Here, we describe a protocol that was employed to quantitatively analyze the binding of annexin A2 (AnxA2) to planar lipid bilayers prepared on the surface of a quartz sensor. This protocol is optimized to obtain robust and reproducible data and includes a detailed step-by-step description. The method can be applied to other membrane-binding proteins and bilayer compositions.

Here, we present an experimental protocol that can be employed to determine the binding affinities and mode of interaction of label-free phospholipid-binding protein annexin A2 with immobilized solid-supported bilayers (SLB) by simultaneously measuring the mass uptake and the viscoelastic properties of the protein annexin A2.

散逸の Microgravimetry 結合タンパク質アネキシン A2 への水晶振動子を用いた固相担脂質膜リン脂質の結合動態を研究するには

The dissipative quartz crystal microbalance technique is a simple and label-free approach to measure simultaneously the mass uptake and viscoelastic ...

Measuring and Interpreting Oxygen Consumption Rates in Whole Fly Head Segments

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the progression of cancer, diabetes, neurodegeneration, and aging to name a few. For instance, changes in mitochondrial activity, the cell&#39;s metabolic powerhouse, have been characterized in many such diseases. Generally, the oxygen consumption rates of mitochondria were considered a reliable readout of mitochondrial activity and measurements in some of these studies were based on isolated mitochondria or cells. However, such conditions may not represent the complexity of a whole tissue. Recently, we have developed a novel method that enables the dynamic measurement of oxygen consumption rates from whole isolated fly heads. By utilizing this method, we have recorded lower oxygen consumption rates of the whole head segment in young versus aged flies. Secondly, we have discovered that lysine deacetylase inhibitors rapidly alter the oxygen consumption in the whole head. Our novel technique may therefore aid in uncovering new properties of various drugs, which may impact metabolic rates. Furthermore, our method may give a better understanding of metabolic behavior in an experimental setup that more closely resembles physiological states.

Measuring alterations in metabolic rates is central to understanding the progression of various diseases and aging. Here, we present a novel technique to measure whole head oxygen consumption that more closely resembles the physiological state and may aid in revealing novel drugs that modify mitochondrial activity.

測定と酸素消費量全体の解釈飛ぶ頭セグメント

Regulated metabolic activity is essential for the normal functioning of living cells. Indeed, altered metabolic activity is causally linked with the ...

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19&#8211;22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers.

ポスト染色法によるポリアクリルアミドゲル中のマンゴータグRNAの蛍光可視化

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, ...

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 ...