Name: absolute quantification of plasma microrna levels in cynomolgus monkeys, using quantitative real-time reverse transcription pcr
Uploaded: 2018-02-12T13:30:00
Description: Watch this Scientific Journal Video about Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR at JoVE.com

本研究は、公共、商業、または非営利セクターの資金調達機関から任意の特定の助成金を受信しませんでした。

すべての実験は動物介護制度と株式会社第一三共使用委員会によって承認されました。
1. サンプル準備
<ol><li>EDTA の 2 K を含むチューブにカニクイザルの大腿静脈から血 (少なくとも 0.5 mL) を収集します。 注: クエン酸とヘパリンです・・・・これらの凝固を抑制するその後 PCR14,15</li>
	<li>氷とコレクションの 2 時間以内に?...

<ol>
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Res. 120 (2), 381-399 (2017).</li><li>D'Alessandra, 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=Circulating+microRNAs+are+new+and+sensitive+biomarkers+of+myocardial+infarction.">Circulating microRNAs are new and sensitive biomarkers of myocardial infarction.</a> Eur Heart J. 31 (22), 2765-2773 (2010).</li><li>Chen, Y., Gelfond, J. A., McManus, L. M., Shireman, P. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Why+the+need+for+qPCR+publication+guidelines?--The+case+for+MIQE.">Why the need for qPCR publication guidelines?--The case for MIQE.</a> Methods. 50 (4), 217-226 (2010).</li><li>Kroh, E. M., Parkin, R. K., Mitchell, P. S., Tewari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+circulating+microRNA+biomarkers+in+plasma+and+serum+using+quantitative+reverse+transcription-PCR+(qRT-PCR).">Analysis of circulating microRNA biomarkers in plasma and serum using quantitative reverse transcription-PCR (qRT-PCR).</a> Methods. 50 (4), 298-301 (2010).</li><li>Thompson, K. 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=Absolute+Measurement+of+Cardiac+Injury-Induced+microRNAs+in+Biofluids+across+Multiple+Test+Sites.">Absolute Measurement of Cardiac Injury-Induced microRNAs in Biofluids across Multiple Test Sites.</a> Toxicol Sci. 154 (1), 115-125 (2016).</li><li>Iguchi, T., Niino, N., Tamai, S., Sakurai, K., Mori, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Analysis+of+Circulating+microRNA+Specific+to+the+Liver,+Heart,+and+Skeletal+Muscle+of+Cynomolgus+Monkeys.">Comprehensive Analysis of Circulating microRNA Specific to the Liver, Heart, and Skeletal Muscle of Cynomolgus Monkeys.</a> Int. J. Toxicol. 36 (3), 220-228 (2017).</li><li>Matsuzaka, 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=Three+novel+serum+biomarkers,+miR-1,+miR-133a,+and+miR-206+for+Limb-girdle+muscular+dystrophy,+Facioscapulohumeral+muscular+dystrophy,+and+Becker+muscular+dystrophy.">Three novel serum biomarkers, miR-1, miR-133a, and miR-206 for Limb-girdle muscular dystrophy, Facioscapulohumeral muscular dystrophy, and Becker muscular dystrophy.</a> Environ. Health. Prev. Med. 19 (6), 452-458 (2014).</li><li>Starkey Lewis, P. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+microRNAs+as+potential+markers+of+human+drug-induced+liver+injury.">Circulating microRNAs as potential markers of human drug-induced liver injury.</a> Hepatology. 54 (5), 1767-1776 (2011).</li><li>Wang, K., 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=Circulating+microRNAs,+potential+biomarkers+for+drug-induced+liver+injury.">Circulating microRNAs, potential biomarkers for drug-induced liver injury.</a> Proc. Natl. Acad. Sci. U. S. 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H., 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=Plasma+processing+conditions+substantially+influence+circulating+microRNA+biomarker+levels.">Plasma processing conditions substantially influence circulating microRNA biomarker levels.</a> PLoS One. 8 (6), e64795 (2013).</li><li>Serafin, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+a+set+of+endogenous+reference+genes+for+miRNA+expression+studies+in+Parkinson's+disease+blood+samples.">Identification of a set of endogenous reference genes for miRNA expression studies in Parkinson's disease blood samples.</a> BMC Res. Notes. 7, 715 (2014).</li><li>Akiyama, H., 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=A+set+of+external+reference+controls/probes+that+enable+quality+assurance+between+different+microarray+platforms.">A set of external reference controls/probes that enable quality assurance between different microarray platforms.</a> Anal. Biochem. 472, 75-83 (2015).</li><li>Kirschner, M. B., van Zandwijk, N., Reid, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+microRNAs:+potential+biomarkers+in+need+of+standardized+reporting.">Cell-free microRNAs: potential biomarkers in need of standardized reporting.</a> Front Genet. 4, 56 (2013).</li><li>Malentacchi, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=SPIDIA-RNA:+second+external+quality+assessment+for+the+pre-analytical+phase+of+blood+samples+used+for+RNA+based+analyses.">SPIDIA-RNA: second external quality assessment for the pre-analytical phase of blood samples used for RNA based analyses.</a> PLoS One. 9 (11), e112293 (2014).</li><li>Sourvinou, I. S., Markou, A., Lianidou, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+circulating+miRNAs+in+plasma:+effect+of+preanalytical+and+analytical+parameters+on+their+isolation+and+stability.">Quantification of circulating miRNAs in plasma: effect of preanalytical and analytical parameters on their isolation and stability.</a> J Mol Diagn. 15 (6), 827-834 (2013).</li><li>Yamaura, Y., Nakajima, M., Takagi, S., Fukami, T., Tsuneyama, K., Yokoi, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+microRNA+profiles+in+rat+models+of+hepatocellular+injury,+cholestasis,+and+steatosis.">Plasma microRNA profiles in rat models of hepatocellular injury, cholestasis, and steatosis.</a> PLoS One. 7 (2), e30250 (2012).</li><li>Kirschner, M. B., Edelman, J. J., Kao, S. C., Vallely, M. P., van Zandwijk, N., Reid, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Impact+of+Hemolysis+on+Cell-Free+microRNA+Biomarkers.">The Impact of Hemolysis on Cell-Free microRNA Biomarkers.</a> Front Genet. 4, 94 (2013).</li><li>Mestdagh, P., 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=High-throughput+stem-loop+RT-qPCR+miRNA+expression+profiling+using+minute+amounts+of+input+RNA.">High-throughput stem-loop RT-qPCR miRNA expression profiling using minute amounts of input RNA.</a> Nucleic Acids Res. 36 (21), e143 (2008).</li><li>Pritchard, C. C., Cheng, H. H., Tewari, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MicroRNA+profiling:+approaches+and+considerations.">MicroRNA profiling: approaches and considerations.</a> Nat. Rev. Genet. 13 (5), 358-369 (2012).</li><li>Moldovan, L., Batte, K. E., Trgovcich, J., Wisler, J., Marsh, C. B., Piper, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methodological+challenges+in+utilizing+miRNAs+as+circulating+biomarkers.">Methodological challenges in utilizing miRNAs as circulating biomarkers.</a> J. Cell. Mol. Med. 18 (3), 371-390 (2014).</li><li>Mangolini, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+and+prognostic+microRNAs+in+the+serum+of+breast+cancer+patients+measured+by+droplet+digital+PCR.">Diagnostic and prognostic microRNAs in the serum of breast cancer patients measured by droplet digital PCR.</a> Biomark. Res. 3, 12 (2015).</li><li>Miotto, E., Saccenti, E., Lupini, L., Callegari, E., Negrini, M., Ferracin, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantification+of+circulating+miRNAs+by+droplet+digital+PCR:+comparison+of+EvaGreen-+and+TaqMan-based+chemistries.">Quantification of circulating miRNAs by droplet digital PCR: comparison of EvaGreen- and TaqMan-based chemistries.</a> Cancer Epidemiol. Biomarkers Prev. 23 (12), 2638-2642 (2014).</li></ol>

私たちの包括的な評価には、個々 のサンプルのばらつきの大きさだったテスト miRNAs の間で非常に異なることを明示するダイナミック レンジの範囲のより厳密な統計的分析が用意されています。これらのバリエーションは、その少量の体液中に起因するかもしれないが、ことこれらのデータ反映生物学的変化だけでなく、技術的な変化に留意。技術的な変化のほとんどは、他の分析プラット...

研究の増加する数の診断と癌の予後のバイオ マーカーとしてマイクロ Rna (Mirna) を探索または監視と非臨床および臨床的研究1,2,3 で他の疾患を検出.定量的リアルタイム逆転写 PCR (RT qPCR) は個々 のターゲットの Mirna を評価するため、この手法はより敏感なマイクロ アレイ4と RNA シーケンス ベースのプラットフォーム5よりも、最も一般的な方法の 1 つであります。一般に、miRNA の表現は、ΔCq 方法6を使用して参照サンプルを基準にして測定されます。このアプローチは、ターゲット遺伝子発現レベルでの生理的変化を調査に適しています。ただし、循環する Mirna の相対的な定量化は、少量のためユーティリティを制限されています。また、技術の変化、異なる所カスタマイズ RT qPCR 実験プロトコル、異なる矛盾につながるかも矛盾に起因するため、さまざまな研究からの結果を比較することは困難異なった調査7。
上記懸念の観点から絶対定量が体液の miRNAs の少量の評価に適してあります。絶対定量法は、対応するターゲット miRNA8と同じシーケンス合成の RNA オリゴヌクレオチドの既知濃度から生成される標準曲線を使用します。保健環境科学研究所 (HESI) ゲノミクス研究会は最近複数のテスト サイトのプラズマ Mirna 絶対測定結果を比較するための包括的な研究を実施しました。結果はことの Mirna 絶対 quantitation のための標準プロトコルを使用して複数のテスト サイト9間で同等の結果をもたらした。本研究では説明されている RT qPCR 測定法は HESI の標準のプロトコルは、複数の miRNA ターゲット、および低式 miRNAs の検出を支援するために中古の増幅の多重分析が含まれていますとほぼ同じです。
本研究では一定量 (200 μ L) EDTA 血漿、血液から調製したが意識したカニクイザルの大腿静脈から収集 (n = 50) 使用10だった。次のプロトコルでは、抽出、miRNA と Rt-qpcr、中古増幅を含む血漿検体の準備のための手順について説明します。もっと重要なは、よく修飾プロセスと組み合わせてターゲット miRNAs サンプルの数量を検証できるように、プロトコルに関する追加の技術情報が、含まれていた。まず、各 miRNA の標準曲線は、生体試料中の定量前にその個々 の検出範囲で検証されました。第二に、現在の方法論の質は、外部コントロール (cel-ミール-238) の Cq 値による総合的に行った。したがって、このプラットフォームでは、さまざまな研究や研究室からの結果を比較するためより有益で信頼性の高いデータが得られます。
8 miRNAs のプロファイルは、代表的な結果としてこのレポートに含まれているここで説明した測定法から。潜在的な安全性バイオ マーカーは、心 (ミール-1、ミール 208a、ミール-208b およびミール 499a) と、齧歯動物および人間3筋 (ミール 133a とミール 206) (ミール 122 とミール 192)、肝臓組織の損傷に関連付けられているこれらの Mirna が提案されています。 11,12,13。

<table border="0" cellpadding="0" cellspacing="0" width="697">
	<tbody>
		<tr height="39">
			<td height="39" style="height:39px;width:216px;">BD Microtainer tube (K2EDTA)</td>
			<td style="width:195px;">Becton, Dickinson and Company</td>
			<td style="width:119px;">365974</td>
			<td style="width:167px;">For blood collection</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Eppendorf PCR Tubes, 0.2 mL</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">0030124359</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Eppendorf Safe-Lock micro test tubes&#160; 1.5 mL</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">0030120086</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Eppendorf Safe-Lock micro test tubes&#160; 2.0 mL</td>
			<td style="width:195px;">Eppendorf</td>
			<td style="width:119px;">0030120094</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Synthetic oligonucleotide</td>
			<td style="width:195px;">Hokkaido System Science</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Individual miRNA (0.2 &#956;mol,HPLC grade)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Tris-EDTA Buffer&#160; (pH 8.0)</td>
			<td style="width:195px;">Nippon Gene</td>
			<td style="width:119px;">314-90021</td>
			<td style="width:167px;">TE buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Buffer RPE</td>
			<td style="width:195px;">QIAGEN</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Contents in miRNeasy mini kit&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Buffer RWT</td>
			<td style="width:195px;">QIAGEN</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Contents in miRNeasy mini kit&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">miRNeasy Mini Kit</td>
			<td style="width:195px;">QIAGEN</td>
			<td style="width:119px;">217004</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nuclease-Free Water&#160;</td>
			<td style="width:195px;">QIAGEN</td>
			<td style="width:119px;">129114</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">QIAzol Lysis Reagent</td>
			<td style="width:195px;">QIAGEN</td>
			<td style="width:119px;">-</td>
			<td style="width:167px;">Contents in miRNeasy mini kit&#160;</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Syn-cel-miR-238-3p miScript miRNA Mimic&#160;</td>
			<td style="width:195px;">QIAGEN</td>
			<td style="width:119px;">219600</td>
			<td style="width:167px;">ID:MSY0000293, 5 nmol</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">SC Adapters</td>
			<td style="width:195px;">TAIGEN Bioscience Corporation</td>
			<td style="width:119px;">S0120</td>
			<td style="width:167px;">For RNA extraction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">VacEZor 36 Complete System</td>
			<td style="width:195px;">TAIGEN Bioscience Corporation</td>
			<td style="width:119px;">M3610</td>
			<td style="width:167px;">For RNA extraction</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">7900HT Fast Real-Time PCR System</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4351405</td>
			<td style="width:167px;">Fast 96-Well Block</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">GeneAmp PCR System 9700</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">9700</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MicroAmp Fast Optical 96-Well Reaction Plate</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4346907</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">MicroAmp Optical Adhesive Film</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4311971</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan Fast Advanced Master Mix</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4444557</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (cel-miR-238-3p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 000248</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-122-5p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 002245</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-133a-3p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 002246</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-1-3p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 002222</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-192-5p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 000491</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-206)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 000510</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-208a-3p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 000511</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-208b-3p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 002290</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Assays (hsa-miR-499a-5p)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4427975</td>
			<td style="width:167px;">Assay ID: 001352</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan MicroRNA Reverse 
			Transcription Kit</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4366597</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">TaqMan PreAmp Master Mix (2&#215;)</td>
			<td style="width:195px;">Thermo Fisher Scientific Inc.</td>
			<td style="width:119px;">4391128</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Chloroform&#160;</td>
			<td style="width:195px;">Wako Pure Chemicals</td>
			<td style="width:119px;">035-02616</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Ethanol (99.5)</td>
			<td style="width:195px;">Wako Pure Chemicals</td>
			<td style="width:119px;">057-00456</td>
			<td style="width:167px;"></td>
		</tr>
	</tbody>
</table>

absolute quantification of plasma microrna levels in cynomolgus monkeys, using quantitative real-time reverse transcription pcr

RT qPCR は個々 のターゲットの Mirna を評価する最も一般的な方法の 1 つです。Mirna のレベルは一般に参照サンプルを基準にして測定されます。このアプローチは、ターゲット遺伝子の表現のレベルの生理学的変化を調べることに適しています。しかしより良い統計解析を用いた絶対定量遺伝子発現レベルの包括的な評価をお勧めします。絶対定量はまだ共通の使用します。このレポートでは、中古増幅の有無 Rt-qpcr を使用してプラズマ miRNA の絶対レベルを測定するためのプロトコルについて説明します。
EDTA 血漿の固定ボリューム (200 μ L) を意識したカニクイザルの大腿静脈から集められた血から調製した (n = 50)。総 RNA は、市販のシステムを使用して抽出しました。プラズマ miRNAs は miRNA 特定フォワード/リバースの PCR プライマーとプローブを含むプローブ ベースの RT qPCR アッセイによって定量化されました。絶対的な定量化のための標準的な曲線は、市販の合成 RNA オリゴヌクレオチドを使用して生成されました。合成 cel-ミール-238 は、正規化と品質評価のための外部コントロールとして使用されました。35 上記サイクル (Cq) 値の定量化を示した miRNAs は qPCR ステップ前にあらかじめ増幅.
検査 8 Mirna の中では、ミール-122、ミール-133a、ミール 192 ミール 1 とミール-206、ミール 499a の低発現量のため中古増幅に必要なに対しあった中古増幅なし検出。ミール 208a とミール 208b 検出されなかった中古増幅後でさえも。サンプル処理効率は、スパイクの cel ミール 238 の Cq 値で評価しました。この測定法の技術的な変化が 3 倍未満と推定され、定量化 (LLOQ) の下限が 102コピー/μ L、検討の miRNAs のほとんどのため。
このプロトコル プラズマ miRNAs の量のよりよい見積もりを提供でき、さまざまな研究から対応するデータの品質評価。体液、中古増幅 miRNAs の低い数字は悪いの検出を向上に役立ちます考慮した Mirna を表明しました。

RT qPCR による miRNA アッセイのワークフロー、品質 assessment 図 1は、qPCR10を使用して血液サンプルからミルナ アッセイのワークフローを示しています。実験の品質は、cel-ミール-238 として外部からの制御を含む、検証できます。これは RNA の抽出の技術の変化を明らかにし、その後 RT qPCR 処理します。本研究...

Watch this Scientific Journal Video about Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR at JoVE.com

Absolute Quantification of Plasma MicroRNA Levels in Cynomolgus Monkeys, Using Quantitative Real-time Reverse Transcription PCR

このレポートでは、中古増幅の有無、定量的リアルタイム逆転写 PCR を用いたプラズマ miRNA の絶対レベルを測定するためのプロトコルについて説明します。このプロトコル プラズマ miRNAs の量のより良い理解とさまざまな研究や研究室から対応するデータの質的評価をことができます。

定量的リアルタイム逆転写 PCR を用いたカニクイザルにおける血中マイクロ Rna の絶対定量

RT-qPCR is one of the most common methods to assess individual target miRNAs. MiRNAs levels are generally measured relative to a reference sample. This ...

The overall goal of this procedure is to measure the absolute levels of Plasma MicroRNAs using Quantitative Real-time Reverse Transcription PCR. This method can help assess the quantity of plasma microRNAs, even if their expression level is low. The main advantage of this technique is that the measured data can be compared with others from different studies or laboratories.The implications of this technique extend to the samples from other species because a standard curve can be generated by using the synthetic RNA or the nucleotide. This method can provide insight into translational research to investigate promising safety biomarkers. First, thaw the frozen plasma samples obtained from the femoral vein of the Cynomolgus Monkeys on ice.Transfer the Lysis reagent in Chloroform on ice for chilling before RNA extraction. Next, add 1, 000 microliters of Lysis reagent to 200 microliters of the sample. And vortex for one minute to ensure proper mixing.Then add five microliters of five nanomolar synthetic Caenorhabditis elegans microRNA and 200 microliters of chloroform to the sample. Vortex the sample for one minute to mix properly. Then, place the sample on ice for two to three minutes.Next, centrifuge the sample at 12, 000 G, for 15 minutes at four degrees Celsius. Next, carefully transfer 650 microliters of the aqueous phase to a new microtube. Then, add 975 microliters of Ethanol to the aqueous phase and pipette several times to ensure complete mixing.Transfer the sample into the corresponding column and adapter and then vacuum dry for three minutes using vacuum manifolds. Then add 200 microliters of Ethanol to the column and vacuum dry for one minute. After vacuum drying, add 800 microliters of RWT Buffer to the column and, again, vacuum dry for two minutes.Then add 800 microliters of RPE Buffer to the column and vacuum dry for two minutes. Add the RPE Buffer twice, followed by vacuum drying. Next, add 300 microliters of Ethanol to the column.After adding Ethanol, vacuum dry for one minute. Then, transfer the column in a new microtube and centrifuge the same at 12, 000 G at room temperature for one minute. After centrifugation, transfer the column to a new microtube and add 50 microliters of Nuclease-free water to it.Let the column stand at room temperature for three minutes and then centrifuge at 8, 000 G at room temperature for one minute. Finally, store the eluate at minus 80 degrees Celsius until use. To prepare a synthetic RNA Oligonucleotide Concentration that corresponds to the target microRNA, dilute the stock solution 10-fold to achieve a working solution with the highest concentration to plot the standard curves.Next, to constitute a multiplex pool of reverse transcription primers, mix equal volumes of the 20-strength primers for target microRNAs. Then, prepare the reverse transcription reaction mix. Then add 10 microliters of reverse transcription reaction mix to five microliters of RNA sample.Mix the two by pipetting several times. Then, incubate the mixture on ice for five minutes. After placing the sample in the Thermocycler, start the cycle.Once the program is over, store the reverse transcribed sample at minus 80 degrees Celsius. To constitute the multiplex assay primer pool, add equal volumes of five microliters of assay primers corresponding to target microRNAs into the tube containing Tris-EDTA buffer in the final volume of 1, 000 microliters. Next, constitute the pre-amplification reaction mix.Once the reaction mix is done, transfer 22.5 microliters of pre-amplification reaction mix to two point five microliters of reversed-transcribed sample. Then, pipette to mix the sample and pre-amplification master mix thoroughly and incubate on ice for five minutes and then leave the PCR tubes in the Thermocycler and start the run. Once the program is over, transfer all the samples at minus 80 degrees Celsius.After thawing the pre-amplified or non-pre-amplified samples, dilute it five-folds with sterile water. To generate the standard curve, do 10-fold serial dilution of the samples derived from synthetic RNA oligonucleotides. Then, constitute the quantitative PCR reaction mix in a tube kept on ice.Then add 18 microliters from the quantitative PCR reaction mix to the Fast Optical 96-Well Reaction Plates. Then add two microliters of the diluted samples into the wells. After sealing the plate with adhesive film, briefly centrifuge the sample at 500 G for 15 seconds.Then start the real-time Thermocycler program. Use a SDS software Version 2.4 that functions with the corresponding real-time thermal cycler to compute the raw copy number of each sample. Then calculate the absolute copy number from the raw copy number using Excel software.Amplification efficiency of miR-122, miR-192, and miR-133a was analyzed by plotting standard curves, which explains the relation between the log concentration versus the quantification cycle for the non-pre-amplified samples. The standard curves for miR-122, miR-192, and miR-133a show a linear relationship between quantification cycles and log concentration of the samples. Next, amplification efficiency of miR-1, miR-499a, and miR-206 was analyzed by plotting standard curves for pre-amplified samples.To analyze the amplification efficiency, the slope was computed using linear regression. The slope in standard curve A shows the absence of specific amplicons at 1, 000 copies per microliter concentration for miR-499a and miR-206. However, the non-specific amplicons were obtained at 1, 000 copies per microliter for miR-1.Here, non-pre-amplified and pre-amplified miR-206 samples were used in duplicates to derive the amplification plots. The quantification cycle values for non-pre-amplified and pre-amplified miR-206 was estimated to be 39.9, plus or minus one point nine and 27.0, plus or minus two point two, respectively, representing almost similar values between the duplicates as shown in the plots. Next, plasma microRNAs obtained from Cynomolgus monkeys were subjected to profiling to compute their absolute values.The dot plots, obtained from microRNA profiling representing their expression levels, shows that miR-122, miR-133a, and miR-192 is detectable without pre-amplification. Whereas, miR-1, miR-206, and miR-499a will require pre-amplification due to low expression levels. After watching this video, you should have a good understanding of how to measure the absolute levels of plasma microRNA, using RT-qPCR.And pre-amplification;it's useful to improve detection with small quantities of microRNAs.

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