Name: circulating microrna quantification using dna-binding dye chemistry and droplet digital pcr
Uploaded: 2016-06-26T14:00:00
Description: Watch this Scientific Journal Video about Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR at JoVE.com

（ - 2010/15、9980ミルのn個当たり5。特別プログラム分子臨床腫瘍）と命令のイタリア省から、大学や癌研究のためのイタリア語協会（AIRC）からMF（MFAG 11676）にし、MNに資金を提供することによってサポートされていますMNへの研究FIRB 2011（プロジェクトRBAPIIBYNP）。

血漿または血清からのマイクロRNAの単離注：血漿および血清調製は、miRNAの定量化を循環関連のステップです。血漿および血清の調製のための好ましい手順はありません。検討する唯一の重要なことは、同じ実験からの全てのサンプルは正確に同じワークフローを使用して処理しなければならないことです。 200μlの血清または血漿から開始します。総RNAは、市販のキットを用いて血清ま?...

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循環するmiRNAは、非常に低濃度で血液中に存在し、血漿および血清試料から抽出することができるRNAの量は低いです。この理由のため、それらは、マイクロアレイおよびRNA配列のような他の技術を用いて定量化することは困難です。また、データの正規化と血液中の内因性の「基準」のmiRNAの存在に合意の一般化が欠如しています。この文脈において、液滴デジタルPCRのような敏感な技術は、...

MicroRNAs (miRNAs) are released into blood circulation by potentially all the cells of the organism, as a consequence of active release or necrotic and apoptotic processes. Cell-free miRNAs have been detected in the bloodstream either as free stable molecules or linked to lipoproteins or enveloped inside exosomes and microvesicles 1-3. They are believed to function as cell-to-cell communicators 4, and their amount changes in the presence of cancer, cardiac disorders or autoimmune diseases 5-7. Their accurate and reproducible quantification is the basis for their evaluation as disease biomarkers. However, for several reasons already described elsewhere 8,9, miRNA quantification in serum or plasma, as well as other body fluids, could be very challenging 10,11. We recently developed a method for the absolute quantification of circulating miRNAs, based on miRNA-specific LNA primers and DNA-binding dye droplet digital PCR (ddPCR) technology 12. This methodology has been applied to the validation of miRNA breast cancer biomarkers 13,14. 
After the partitioning of each reverse-transcribed miRNA molecule inside a nanoliter-sized droplet, it is possible to count the copy number of each miRNA in each sample, basically counting the number of green, and therefore positive, fluorescent droplets. As soon as a PCR reaction occurs, a positive count is achieved, without the need to establish a standard curve or taking PCR efficiency into account in target amount calculation, as it happens with quantitative RT-PCR (RT-qPCR). In addition, ddPCR proved to be more sensitive and accurate than RT-qPCR in circulating miRNA quantification 15. In this article we present the detailed protocol of this methodology, discussing the most relevant steps andspecifically considering serum and plasma clinical samples.

<table >
	
		
		
		
		
	
	<tbody>
		<tr >
			<td >miRNeasy Mini Kit</td>
			<td >Qiagen</td>
			<td >217004</td>
			<td >Columns for total RNA, including miRNA, extraction from serum/plasma</td>
		</tr>
		<tr >
			<td >100 nmole RNA oligo Cel-miR-39-3p</td>
			<td >Integrated DNA Technologies</td>
			<td >Custom</td>
			<td >Sequence: UCACCGGGUGUAAAUCAGCUUG</td>
		</tr>
		<tr >
			<td >Universal cDNA synthesis kit II, 8-64 rxns</td>
			<td >Exiqon</td>
			<td >203301</td>
			<td >Kit for microRNA reverse transcription</td>
		</tr>
		<tr >
			<td >MicroRNA LNA PCR primer set&#160;</td>
			<td >Exiqon</td>
			<td >204000-206xxx and 2100000-21xxxxx</td>
			<td >Primers for miRNA amplification inside droplets</td>
		</tr>
		<tr >
			<td >QX200 droplet generator</td>
			<td >BioRad</td>
			<td >186-4002</td>
			<td >Instrument used for droplet reading</td>
		</tr>
		<tr >
			<td >QX200 droplet reader</td>
			<td >BioRad</td>
			<td >186-4003</td>
			<td >Instrument used for droplet generation</td>
		</tr>
		<tr >
			<td >QuantaSoft software</td>
			<td >BioRad</td>
			<td >186-3007</td>
			<td >Software for data collection and analysis</td>
		</tr>
		<tr >
			<td >PX1 PCR plate sealer</td>
			<td >BioRad</td>
			<td >181-4000</td>
			<td >Plate sealer</td>
		</tr>
		<tr >
			<td >DG8 droplet generator cartridges and gaskets</td>
			<td >BioRad</td>
			<td >186-4008</td>
			<td >Cartridges used to mix sample and oil to generate droplets</td>
		</tr>
		<tr >
			<td >QX200 ddPCR EvaGreen supermix</td>
			<td >BioRad</td>
			<td >186-4033/36</td>
			<td >PCR supermix</td>
		</tr>
		<tr >
			<td >QX200 droplet generator oil for EvaGreen dye</td>
			<td >BioRad</td>
			<td >186-4005</td>
			<td >Oil for droplet generation</td>
		</tr>
	</tbody>
</table>

circulating microrna quantification using dna-binding dye chemistry and droplet digital pcr

マイクロRNA（miRNAは）（無細胞の）循環血液流に細胞から放出されます。循環中の特定のマイクロRNAの量は、疾患状態に連結され、疾患のバイオマーカーとして使用する可能性を有するされています。デジタルPCR技術染料系化学を用いて、液滴マイクロRNA定量を循環させるための高感度で正確な方法が最近開発されました。具体的には、ロックされた核酸（LNA）を使用することは、特定のmiRNAの絶対的な定量化を得ることができる互換性のある液滴デジタルPCRシステムにおいて緑色蛍光DNA結合色素とのmiRNA特異的プライマーをベース。ここでは、そのような血漿および血清などの生物学的流体、中のmiRNAの量を評価するためにこの技術を実行方法について説明し、実行可能かつ効果的です。

血漿または血清の1ml当たりの特定のmiRNAの絶対量は、緑色蛍光DNA結合色素を用いて測定し、デジタルPCR技術滴することができる。 図1は、最終的なmiRNAの濃度を決定する正の液滴の選択、（コピー/μLのプロセスを示します）解析ソフトウェアにより計算増幅反応です。血液中の各miRNAの量が他のものよりも豊富ないくつかのmiRNA種であること、非常に異なっ...

Watch this Scientific Journal Video about Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR at JoVE.com

Circulating MicroRNA Quantification Using DNA-binding Dye Chemistry and Droplet Digital PCR

A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

DNA結合色素化学と液滴デジタルPCRを使用したマイクロRNA定量循環

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been ...

Research

JoVE Journal

Bioengineering

High Throughput MicroRNA Profiling: Optimized Multiplex qRT-PCR at Nanoliter Scale on the Fluidigm Dynamic ArrayTM IFCs

ハイスループットマイクロRNAプロファイリング：最適化されたマルチプレックスフリューダイナミックArrayTMのIFCSでナノリッタースケールでの定量RT - PCR

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the ...

Fluorescence detection methods for microfluidic droplet platforms

マイクロ流体液滴のプラットフォームのための蛍光検出法

The development of microfluidic platforms for performing chemistry and biology has in large part been driven by a range of potential benefits that ...

Detection and Isolation of Circulating Melanoma Cells using Photoacoustic Flowmetry

光音響流量測定法を用いて循環メラノーマ細胞の検出と分離

Circulating tumor cells (CTCs) are those cells that have separated from a macroscopic tumor and spread through the blood and lymph systems to seed ...

Diagnosis of Neoplasia in Barrett&#8217;s Esophagus using Vital-dye Enhanced Fluorescence Imaging

Diagnosis of Neoplasia in BarrettÃƒÂ¢Ã¢â€šÂ¬Ã¢â€žÂ¢s Esophagus using Vital-dye Enhanced Fluorescence Imaging

バイタル色素増強蛍光イメージングを使用して、バレット食道腫瘍の診断

The ability to differentiate benign metaplasia in Barrett&#8217;s Esophagus (BE) from neoplasia in vivo remains difficult as both tissue types can be flat ...

Quantification and Size-profiling of Extracellular Vesicles Using Tunable Resistive Pulse Sensing

調整可能な抵抗パルスセンシングを使用して定量化し、細胞外小胞の大きさ、プロファイリング

Extracellular vesicles (EVs), including &#8216;microvesicles&#8217; and &#8216;exosomes&#8217;, are highly abundant in bodily fluids. Recent years have ...

Multicolor Fluorescence Detection for Droplet Microfluidics Using Optical Fibers

光ファイバを用いて、液滴マイクロフルイディクスのための多色蛍光検出

Fluorescence assays are the most common readouts used in droplet microfluidics due to their bright signals and fast time response. Applications such as ...

A Method to Estimate Cadaveric Femur Cortical Strains During Fracture Testing Using Digital Image Correlation

破壊を使用してデジタル画像相関法を試験中に死体大腿骨皮質系統を推定する方法

This protocol describes the method using digital image correlation to estimate cortical strain from high speed video images of the cadaveric femoral ...

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy (DHM)

Quantifying Microorganisms at Low Concentrations Using Digital Holographic Microscopy DHM

デジタル ホログラフィー顕微鏡 (DHM) を用いた低濃度で微生物の定量化

Accurately detecting and counting sparse bacterial samples has many applications in the food, beverage, and pharmaceutical processing industries, in ...

A Droplet-Based Microfluidic Approach and Microsphere-PCR Amplification for Single-Stranded DNA Amplicons

マイクロ液滴手法と一本鎖 DNA 産物の微粒子に PCR 増幅

Droplet-based microfluidics enable the reliable production of homogeneous microspheres in the microfluidic channel, providing controlled size and ...

Bulk Droplet Vitrification for Primary Hepatocyte Preservation

原発性肝細胞保存のためのバルク液滴ガラス化

Vitrification is a promising ice-free alternative for classic slow-freezing (at approximately 1 &#176;C/min) cryopreservation of biological samples. ...