Name: a method for measuring rna n6-methyladenosine modifications in cells and tissues
Uploaded: 2016-12-05T13:00:00.000+00:00
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Description: Watch this Scientific Journal Video about A Method for Measuring RNA N6-methyladenosine Modifications in Cells and Tissues at JoVE.com

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. 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=FTO+modulates+fibrogenic+responses+in+obstructive+nephropathy.">FTO modulates fibrogenic responses in obstructive nephropathy.</a> Sci Rep. 6, 18874 (2016).</li><li>Li, X., 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=Transcriptome-wide+mapping+reveals+reversible+and+dynamic+N-methyladenosine+methylome.">Transcriptome-wide mapping reveals reversible and dynamic N-methyladenosine methylome.</a> Nat Chem Biol. , (2016).</li><li>Sanschagrin, S., Yergeau, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Next-generation+sequencing+of+16S+ribosomal+RNA+gene+amplicons.">Next-generation sequencing of 16S ribosomal RNA gene amplicons.</a> J Vis Exp. (90), (2014).</li><li>Kukutla, P., Steritz, M., Xu, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Depletion+of+ribosomal+RNA+for+mosquito+gut+metagenomic+RNA-seq.">Depletion of ribosomal RNA for mosquito gut metagenomic RNA-seq.</a> J Vis Exp. (74), (2013).</li><li>Mishima, E., 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=Immuno-Northern+Blotting:+Detection+of+RNA+Modifications+by+Using+Antibodies+against+Modified+Nucleosides.">Immuno-Northern Blotting: Detection of RNA Modifications by Using Antibodies against Modified Nucleosides.</a> PLoS One. 10 (11), 0143756 (2015).</li><li>Klungland, A., Dahl, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamic+RNA+modifications+in+disease.">Dynamic RNA modifications in disease.</a> Curr Opin Genet Dev. 26, 47-52 (2014).</li></ol>

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><tbody><tr><td>RNaseZap solution</td><td>Ambion</td><td>AM9782</td><td>Protocol 1.1</td></tr><tr><td>TRI Reagent solution</td><td>Ambion</td><td>AM9738</td><td>Protocol 1.2.1.1</td></tr><tr><td>1-Bromo-3-chloropropane (BCP)</td><td>Sigma</td><td>B9673</td><td>Protocol 1.2.1.3</td></tr><tr><td>Ethanol</td><td>JTbaker</td><td>8006</td><td>Protocol 1.2.1.3</td></tr><tr><td>Isopropanol</td><td>Sigma</td><td>I9516</td><td>Protocol 1.2.1.3</td></tr><tr><td>GenElute mRNA Miniprep Kit</td><td>Sigma</td><td>MRN70</td><td>Protocol 1.2.2.1</td></tr><tr><td>Glycogen</td><td>Ambion</td><td>AM9510</td><td>Protocol 1.2.2.2</td></tr><tr><td>Sodium acetate</td><td>Fluka</td><td>71183</td><td>Protocol 1.2.2.2</td></tr><tr><td>Nuclease-free water</td><td>Ambion</td><td>AM9930</td><td>Protocol 1.2.2.6</td></tr><tr><td>DEPC</td><td>Sigma</td><td>D5758</td><td>Protocol 2.1.1</td></tr><tr><td>Agarose</td><td>JT Baker</td><td>A426</td><td>Protocol 2.1.2</td></tr><tr><td>MOPS</td><td>Sigma</td><td>M1254</td><td>Protocol 2.1.3</td></tr><tr><td>37% formaldehyde Solution</td><td>Sigma</td><td>F8775</td><td>Protocol 2.1.3</td></tr><tr><td>EDTA, Disodium Salt</td><td>JT Baker</td><td>8993</td><td>Protocol 2.1.3 10x MOPS buffer</td></tr><tr><td>formamide</td><td>Sigma</td><td>F7503</td><td>Protocol 2.2.1</td></tr><tr><td>RNA Millennium Marker</td><td>Ambion</td><td>AM7150</td><td>Protocol 2.2.2</td></tr><tr><td>Ethidium Bromide</td><td>Amresco</td><td>X328</td><td>Protocol 2.2.5</td></tr><tr><td>Ficoll PM400</td><td>GE Healthcare</td><td>17-0300-10</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Bromophenol blue</td><td>Sigma</td><td>114391</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Xylene Cyanol FF</td><td>Sigma</td><td>X4126</td><td>Protocol 2.2.5 tracking dye</td></tr><tr><td>Sodium chloride dihydrate</td><td>JT Baker</td><td>3624</td><td>Protocol 2.4.2 20x SSC buffer</td></tr><tr><td>Trisodium citrate</td><td>Sigma</td><td>S1804</td><td>Protocol 2.4.2 20x SSC buffer</td></tr><tr><td>Hybond Blotting Paper</td><td>GE Healthcare</td><td>RPN6101M</td><td>Protocol 2.4.4</td></tr><tr><td>GE Hybond-N+ membrane</td><td>GE Healthcare</td><td>RPN303B</td><td>Protocol 2.4.6</td></tr><tr><td>Stratalinker UV Crosslinker 2400</td><td>Stratagene</td><td>400075</td><td>Protocol 3.1.2</td></tr><tr><td>Gel Catcher 1500</td><td>ANT Technology</td><td>Gel Catcher 1500</td><td>Protocol 3.1.5</td></tr><tr><td>Anti-m&lt;sup&gt;6&lt;/sup&gt;A (N&lt;sup&gt;6&lt;/sup&gt;-methyladenosine)</td><td>Synaptic Systems</td><td>202003</td><td>Protocol 3.2.3</td></tr><tr><td>Amersham ECL Anti-Rabbit IgG, HRP-linkd whole Ab</td><td>GE Healthcare</td><td>NA934</td><td>Protocol 3.2.5</td></tr><tr><td>ChemiDoc MP System</td><td>BIO-RAD</td><td>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.bdigitaluss.remotexs.co/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.bdigitaluss.remotexs.co/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 ...

a-method-for-measuring-rna-n6-methyladenosine-modifications-cells

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

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

10.9K 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 In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved N6-isopentenyladenosine modifications occurs at the A37 position in a subset of tRNAs. This modification improves translation efficiency and fidelity by increasing the affinity of the tRNA for the ribosome. Mutation of enzymes responsible for this modification in eukaryotes are associated with several disease states, including mitochondrial dysfunction and cancer. Therefore, understanding the substrate specificity and biochemical activities of these enzymes is important for understanding of normal and pathologic eukaryotic biology. A diverse array of methods has been employed to characterize i6A modifications. Herein is described a direct approach for the detection of isopentenylation by Mod5. This method utilizes incubation of RNAs with a recombinant isopentenyl transferase, followed by RNase T1 digestion, and 1-dimensional gel electrophoresis analysis to detect i6A modifications. In addition, the potential adaptability of this protocol to characterize other RNA-modifying enzymes is discussed.

An In Vitro Assay to Detect tRNA-Isopentenyl Transferase Activity

Here, we describe a protocol for the biochemical characterization of the yeast RNA-modifying enzyme, Mod5, and discuss how this protocol could be applied to other RNA-modifying enzymes.

TRNA イソペンテニル トランスフェラーゼ活性を検出する体外の試金

N6-isopentenyladenosine RNA modifications are functionally diverse and highly conserved among prokaryotes and eukaryotes. One of the most highly conserved ...

生化学

Antibody-Free Assay for RNA Methyltransferase Activity Analysis

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and especially methylations, has re-emerged due to the important roles played by the enzymes that write and erase them in biological processes relevant to disease and cancer. Here, a sensitive in vitro assay for accurate analysis of RNA methylation writer activity on synthetic or in vitro transcribed RNAs is provided. This assay uses a tritiated form of S-adenosyl-methionine, resulting in direct labeling of methylated RNA with tritium. The low energy of tritium radiation makes the method safe, and pre-existing methods of tritium signal amplification, make it possible to quantify and to visualize the methylated RNA without the use of antibodies, which are commonly prone to artifacts. While this method is written for RNA methylation, few tweaks make it applicable to the study of other RNA modifications that can be radioactively labeled, such as RNA acetylation with 14C acetyl coenzyme A. Overall, this assay allows to quickly assess RNA methylation conditions, inhibition with small molecule inhibitors, or the effect of RNA or enzyme mutants, and provides a powerful tool to validate and expand results obtained in cells.

Here, an antibody-free in vitro assay for direct analysis of methyltransferase activity on synthetic or in vitro transcribed RNA is described.

RNAメチルトランスフェラーゼ活性分析のための抗体フリーアッセイ

There are more than 100 chemically distinct modifications of RNA, two thirds of which consist of methylations. Interest in RNA modifications, and ...

遺伝学

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

小型RNA-seqの改善:バイアスの減少と2'-O-メチルRNAの検出の改善

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and sequencing (MeRIP-seq) is a method that aims to enrich for methylated RNA and ultimately identify modified transcripts. Briefly, sonicated RNA is incubated with an antibody for 5-methylated cytosines and precipitated with the assistance of protein G beads. The enriched fragments are then sequenced and the potential methylation sites are mapped based on the distribution of the reads and peak detection. MeRIP can be applied to any organism, as it does not require any prior sequence or modifying enzyme knowledge. In addition, besides fragmentation, RNA is not subjected to any other chemical or temperature treatment. However, MeRIP-seq does not provide single-nucleotide prediction of the methylation site as other methods do, although the methylated area can be narrowed down to a few nucleotides. The use of different modification-specific antibodies allows MeRIP to be adjusted for the different base modifications present on RNA, expanding the possible applications of this method.

Methylated RNA Immunoprecipitation Assay to Study m5C Modification in Arabidopsis

The methylated RNA immunoprecipitation assay is an antibody-based method used to enrich for methylated RNA fragments. Coupled with deep sequencing, it leads to the identification of transcripts carrying the m5C modification.

シロイヌナズナのm5C修飾を研究するメチル化RNA免疫沈降アッセイ

Secondary base modifications on RNA, such as m5C, affect the structure and function of the modified RNA molecules. Methylated RNA Immunoprecipitation and ...

2D-HELS MS Seq: A General LC-MS-Based Method for Direct and de novo Sequencing of RNA Mixtures with Different Nucleotide Modifications

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a complementary DNA (cDNA) intermediate. However, such approaches are rarely applied as a de novo RNA sequencing method, but used mainly as a tool that can assist in quality assurance for confirming known sequences of purified single-stranded RNA samples. Recently, we developed&#160;a direct RNA sequencing method by integrating a 2-dimensional mass-retention time hydrophobic end-labeling strategy into MS-based sequencing (2D-HELS MS Seq). This method is capable of accurately sequencing single RNA sequences as well as mixtures containing up to 12 distinct RNA sequences. In addition to the four canonical ribonucleotides (A, C, G, and U), the method has the capacity to sequence RNA oligonucleotides containing modified nucleotides. This is possible because the modified nucleobase either has an intrinsically unique mass that can help in its identification and its location in the RNA sequence, or can be converted into a product with a unique mass. In this study, we have used RNA, incorporating two representative modified nucleotides&#160;(pseudouridine (&#936;) and 5-methylcytosine (m5C)), to illustrate the application of the method for the de novo sequencing of a single RNA oligonucleotide&#160;as well as a mixture of RNA oligonucleotides, each with a different sequence and/or modified nucleotides. The procedures and protocols described here to sequence these model RNAs will be applicable to other short RNA samples (&#60;35 nt) when using a standard high-resolution LC-MS system, and can also be used for&#160;sequence verification of modified therapeutic RNA oligonucleotides.&#160;In the future, with the development of more robust algorithms and with better instruments, this method could allow sequencing of more complex biological samples.&#160;

<meta charset="utf8"></head><body>2D-HELS MS Seq:異なるヌクレオチド修飾を持つRNA混合物の直接およびde novoシーケンシングのための一般的なLC-MSベースの方法

Here, we describe a detailed protocol for an LC-MS-based sequencing method that can be used as a direct method to sequence short RNA (&#60;35 nt per run) without a cDNA intermediate, and as a general method to sequence different nucleotide modifications in a single study at single-base precision.

2D-HELS MS Seq:異なるヌクレオチド修飾を持つRNA混合物の直接およびde novoシーケンシングのための一般的なLC-MSベースの方法

Mass spectrometry (MS)-based sequencing approaches have been shown to be useful in&#160;direct&#160;sequencing&#160;RNA without the need for a ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

核磁気共鳴分光法によるアルギニンメチロームの探索

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

がん研究

Plant Sample Preparation for Nucleoside/Nucleotide Content Measurement with An HPLC-MS/MS

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which are involved in many cell activities. Here, we describe a rapid and reliable method for the absolute qualification of nucleoside/nucleotide contents in plants. Briefly, 100 mg of homogenized plant material was extracted with 1 mL of extraction buffer (methanol, acetonitrile, and water at a ratio of 2:2:1). Later, the sample was concentrated five times in a freeze dryer and then injected into an HPLC-MS/MS. Nucleotides were separated on a porous graphitic carbon (PGC) column and nucleosides were separated on a C18 column. The mass transitions of each nucleoside and nucleotide were monitored by mass spectrometry. The contents of the nucleosides and nucleotides were quantified against their external standards (ESTDs). Using this method, therefore, researchers can easily quantify nucleosides/nucleotides in different plants.

A precise and reproducible method for in vivo nucleosides/nucleotides quantification in plants is described here. This method employs an HPLC-MS/MS.

HPLC-MS/MSによるヌクレオシド/ヌクレオチド含有量測定のための植物サンプル調製

Nucleosides/nucleotides are building blocks of nucleic acids, parts of cosubstrates and coenzymes, cell signaling molecules, and energy carriers, which ...

DNAzyme-dependent Analysis of rRNA 2&#8217;-O-Methylation

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in higher eukaryotes may promiscuously recognize other RNA species and 2&#8217;-O-methylate multiple targets. Here, we provide step-by-step guide for the fast and non-expensive analysis of the site-specific 2&#8217;-O-methylation using a well-established method employing short DNA oligonucleotides called DNAzymes. These DNA fragments contain catalytic sequences which cleave RNA at specific consensus positions, as well as variable homology arms directing DNAzyme to its RNA targets. DNAzyme activity is inhibited by 2-&#8217;O-methylation of the nucleotide adjacent to the cleavage site in the RNA. Thus, DNAzymes, limited only by the consensus of the cleaved sequence, are perfect tools for the quick analysis of snoRNA-mediated RNA 2&#8217;-O-methylation. We analyzed snoRNA snR13- and snR47-guided 2&#8217;-O-methylation of 25S ribosomal RNA in Saccharomyces cerevisiae to demonstrate the simplicity of the technique and to provide a detailed protocol for the DNAzyme-dependent assay.

Here we present a protocol for DNAzyme-dependent cleavage of RNA. This enables fast and site-dependent analysis of RNA 2&#8217;-O-methylation. This approach can be used for the preliminary or major assessment of snoRNA activity.

rRNA 2'-O-メチル化のDNAzyme依存性分析

Guide box C/D small nucleolar RNAs (snoRNAs) catalyze 2&#8217;-O-methylation of ribosomal and small nuclear RNA. However, a large number of snoRNA in ...

発生生物学

Characterizing RNA Modifications in Single Neurons Using Mass Spectrometry

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous system (CNS). Recent evidence has linked specific neuronal RNA modifications to learning and memory paradigms. Unfortunately, conventional methods for the detection of these epitranscriptomic features are only capable of characterizing highly abundant RNA modifications in bulk tissues, precluding the assessment of unique PTM profiles that may exist for individual neurons within the activated behavioral circuits. In this protocol, an approach is described&#8212;single-neuron RNA modification analysis by mass spectrometry (SNRMA-MS)&#8212;to simultaneously detect and quantify numerous modified ribonucleosides in single neurons. The approach is validated using individual neurons of the marine mollusk, Aplysia californica, beginning with surgical isolation and enzymatic treatment of major CNS ganglia to expose neuron cell bodies, followed by manual single-neuron isolation using sharp needles and a micropipette. Next, mechanical and thermal treatment of the sample in a small volume of buffer is done to liberate RNA from an individual cell for subsequent RNA digestion. Modified nucleosides are then identified and quantified using an optimized liquid chromatography-mass spectrometry method. SNRMA-MS is employed to establish RNA modification patterns for single, identified neurons from A. californica that have known morphologies and functions. Examples of qualitative and quantitative SNRMA-MS are presented that highlight the heterogeneous distribution of RNA modifications across individual neurons in neuronal networks.

Post-transcriptional modifications of RNA represent an understudied layer of translation regulation that has recently been linked to central nervous system plasticity. Here, sample preparation and liquid chromatography-tandem mass spectrometry approach is described for simultaneous characterization of numerous RNA modifications in single neurons.

質量分析法を用いた単一ニューロンにおけるRNA修飾の特性評価

Post-transcriptional modifications (PTMs) of RNA represent an understudied mechanism involved in the regulation of translation in the central nervous ...

RNAの測定方法

要約

さらに動画を探す

この動画の章

TRNA イソペンテニルトランスフェラーゼ活性を検出する体外の試金

RNAメチルトランスフェラーゼ活性分析のための抗体フリーアッセイ

小型RNA-seqの改善:バイアスの減少と2'-O-メチルRNAの検出の改善

シロイヌナズナの^m5C修飾を研究するメチル化RNA免疫沈降アッセイ

2D-HELS MS Seq:異なるヌクレオチド修飾を持つRNA混合物の直接およびde novoシーケンシングのための一般的なLC-MSベースの方法

2D-HELS MS Seq:異なるヌクレオチド修飾を持つRNA混合物の直接およびde novoシーケンシングのための一般的なLC-MSベースの方法

核磁気共鳴分光法によるアルギニンメチロームの探索

HPLC-MS/MSによるヌクレオシド/ヌクレオチド含有量測定のための植物サンプル調製

rRNA 2'-O-メチル化のDNAzyme依存性分析

質量分析法を用いた単一ニューロンにおけるRNA修飾の特性評価

RNAの測定方法

要約

さらに動画を探す

この動画の章

TRNA イソペンテニル トランスフェラーゼ活性を検出する体外の試金

RNAメチルトランスフェラーゼ活性分析のための抗体フリーアッセイ

小型RNA-seqの改善:バイアスの減少と2'-O-メチルRNAの検出の改善

シロイヌナズナのm5C修飾を研究するメチル化RNA免疫沈降アッセイ

2D-HELS MS Seq:異なるヌクレオチド修飾を持つRNA混合物の直接およびde novoシーケンシングのための一般的なLC-MSベースの方法

2D-HELS MS Seq:異なるヌクレオチド修飾を持つRNA混合物の直接およびde novoシーケンシングのための一般的なLC-MSベースの方法

核磁気共鳴分光法によるアルギニンメチロームの探索

HPLC-MS/MSによるヌクレオシド/ヌクレオチド含有量測定のための植物サンプル調製

rRNA 2'-O-メチル化のDNAzyme依存性分析

質量分析法を用いた単一ニューロンにおけるRNA修飾の特性評価

TRNA イソペンテニルトランスフェラーゼ活性を検出する体外の試金

シロイヌナズナの^m5C修飾を研究するメチル化RNA免疫沈降アッセイ