Name: circulating microrna quantification using dna-binding dye chemistry and droplet digital pcr
Uploaded: 2016-06-26T14:00:00.000+00:00
Duration: 7 min 37 s
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たは血漿から単離することができます。 1.（miRNAを含む）全RNAのためのプロトコルの単離<ol><li>氷上で解凍血清/血漿サンプル。 </li><li> 200μlの血清/血漿に溶解試薬 ​​（ 例えば 、のQIAzol）の1ミリリットルを追加します。 </li><li>ボルテックスで混合し、室温で5分間、ベンチのホモジネートを含むチューブを置きます。 </li><li> Cから合成miRNA CEL-のmiR-39-3pの4.16 nMの溶液を3μLを加えますエレガンス 。 </li><li> 200μlのクロロホルムを追加します。 15秒間激しくチューブを振ります。目を配置します2分間室温でベンチ上の電子管。 </li><li>相分離を得るために4℃で12,000×gで15分間遠心操作：上部の水相は、RNAが含まれています。 </li><li>任意の白相間材料の移動を防ぐため、約700μlを、新しいチューブに水相を転送します。 </li><li> 100％エタノールの1ミリリットルを加え、反転により混合します。 </li><li>最大700μlのサンプル2 mlのコレクションチューブ上に配置されたミニスピンカラムにピペットで。 15秒間12,000×gで蓋と遠心分離機を閉じます。フロースルーを捨てます。 </li><li>残りのサンプルを使用して、この手順を繰り返します。 </li><li>ミニスピンカラムに700μlのBuffer RWTを追加し、カラムを洗浄するために12,000×gで15秒間蓋と遠心分離機を閉じます。フロースルーを捨てます。 </li><li>ミニスピンカラムにピペットで500μlのBuffer RPE。 12,000 rpmで15秒間蓋と遠心分離機を閉じます。フロースルーを捨てます。もう一度この手順を繰り返します。 </li><li>スピンカラムを乾燥させるために2分間フルスピードでミニスピンカラムを遠心残留エタノールからの膜。 </li><li>カラムのメンブレン上に新しい1.5 mlコレクションチューブとピペット35μlのRNaseフリー水にミニスピンカラムを移します。 </li><li> RNAを溶出するために12,000×gで1分間遠心します。 </li><li> -80℃でRNAを保管してください。 注：RNAの濃度を正確に決定することができないので、入力された量の尺度として固定ボリュームを使用します。 </li></ol> 2.マイクロRNA逆転写<ol><li> 5×反応緩衝液、ヌクレアーゼフリー水：逆転写（RT）試薬ミックス成分を解凍。氷の上管と場所を反転穏やかに混合します。使用直前に、冷凍庫から酵素混合物を除去し、氷の上に置きます。すべての試薬をスピンダウン。 </li><li> 表1に記載されているように氷上でRT試薬ミックスを調製する。少なくとも10％超えるミックスを調製します。 </li><li>ピペット操作によって反応をミックスし、その後スピンダウン。 </li><li> 42℃で60分間インキュベートします。 </li><li> reverを熱不活性化95℃で5分間SE転写酵素。 </li><li>直ちに4℃に冷却します。 </li><li> -20℃でのcDNAを保管してください。 </li></ol> 3. cDNAを希釈<ol><li>ヌクレアーゼを含まない水で計画ddPCR反応に必要なcDNAテンプレートの量を希釈します。午前1時50分と1の間のcDNA反応を希釈水で500、標的miRNAの豊富さに依存します。 -20°Cで希釈したcDNAを保管してください。希釈されたcDNAを、-20℃で少なくとも3ヶ月間安定であることが判明しました。 </li></ol> 4.ドロップレットの生成とPCR 注意：小滴の生成は、一度に8個のサンプルで実行する必要があります。技術的反復が原因12,15 .Aテンプレートなしのコントロール（NTC）サンプルは、すべてのプレートで実行する必要があり、この技術の高い再現性に、それぞれ異なるddPCR条件のために、必要とされていません。 <ol><li>解凍し、マスターミックスを平衡化（ 例えば 、EvaGreenマスターミックス）RTで、マイクロRNAプライマーセットおよびcDNA。 </li><li>マスターミックス番目のミックスoroughlyチューブを数回反転させることによって。すべての試薬をスピンダウン。 </li><li> 表2に記載されているようにddPCRミックスを調製する。複数のddPCR反応が行われた場合、ddPCRが作業-溶液を混合用意。少なくとも10％超えるミックスを調製します。 </li><li>組み立てた後、十分に混合するとddPCRミックスをスピンダウン。 </li><li> ddPCR12μlの96ウェルPCRプレートまたはPCRチューブに混合分配。 </li><li>ウェル/各チューブに希釈したcDNA鋳型の8μlを添加します。 </li><li>ホルダーに液滴発生器のカートリッジを挿入します。 </li><li>空気を回避しながら、液滴発生器のカートリッジのサンプルウェルに移し、各調製した試料を20μl（中段）、慎重には、井戸の底に泡。 </li><li>液滴発生器油の70μlの各油井（下段）を入力します。 </li><li>カートリッジホルダーの上にガスケットをフックし、液滴発生器に挿入します。 </li><li>メーカーの広報に係る液滴生成を開始するためにふたを閉めotocol。液滴生成​​が​​完了したら、ふたを開け、使い捨てガスケットを取り外します。カートリッジの上部ウェルは液滴を含んでいます。 </li><li> 96ウェルPCRプレートの単一の列に8トップウェル（液滴）の内容のピペットでゆっくりとスムーズに40μlの。 </li><li>直ちに蒸発を避けるために、液滴を移送した後、ホイルでPCRプレートを密封します。液滴リーダーに針と互換性のある穿刺可能なホイルプレートシールを使用してください。 </li><li>プレートを封止し、30分以内に熱サイクル（PCR）を開始します。 </li><li> 表3に記載のサイクリングプロトコルを実行します。 </li></ol> 5.ドロップレット読書<ol><li>液滴リーダーの電源をオンにします。 </li><li>液滴リーダーにサーマルサイクラーからプレートを移動します。 </li><li>プレートホルダーのベースにPCR後の液滴を含む96ウェルPCRプレートを置きます。 </li><li> PCRプレート上にプレートホルダーの上部に配置します。しっかりと両方のリリースタブを押してくださいダウンホルダーにPCRプレートを固定します。 </li><li>システムのPCからソフトウェアを起動します。 <ol><li>実験（絶対定量化）を定義するために、セットアップをクリックした後の液滴の読み取りを開始するには[実行]をクリックします。 </li><li>液滴の読み取りが完了すると、データを開き、分析するためのボタンを「分析」をクリックします。 </li><li>正の滴（なげなわツール）（ 図1B）を選択するために、解析ソフトウェアで2D振幅プロットを使用してください。 </li><li>正と総滴数を確認するには、[イベント]タブを使用します。 18000の総数 - 21,000液滴は、通常probeless ddPCRで達成されます。 </li><li>正の液滴を選択したら、集中タブからの輸出の.csvオプションを使用して、miRNAの濃度値をエクスポートします。 </li></ol></li></ol>

<ol>
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The authors have nothing to disclose.

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&amp;nbsp;</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 ...

circulating-microrna-quantification-using-dna-binding-dye-chemistry

Universidad de Cartagena UDEC

Research

JoVE Journal

Bioengineering

8.6K ビュー.  University of Bologna. A sensitive and accurate method for cell-free microRNAs quantification using a dye-based chemistry and droplet digital PCR technology is described.

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

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

The broad involvement of miRNAs in critical processes underlying development, tissue homoeostasis and disease has led to a surging interest among the research and pharmaceutical communities. To study miRNAs, it is essential that the quantification of microRNA levels is accurate and robust. By comparing wild-type to small RNA deficient mouse embryonic stem cells (mESC), we revealed a lack of accuracy and robustness in previous published multiplex qRT-PCR techniques. Here, we describe an optimized method, including purifying away excessive primers from previous multiplex steps before singleplex real time detection, which dramatically increases the accuracy and robustness of the technique. Furthermore, we explain how performing the technique on a microfluidic chip at nanoliter volumes significantly reduces reagent costs and permits time effective high throughput miRNA expression profiling.

Here we describe an optimized multiplex reverse transcriptase quantitative PCR (qRT-PCR) protocol in combination with a microfluidic platform as a cost and time effective high-throughput screening tool for microRNA (miRNA) expression levels, especially when working with limited amounts of sample.

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 ...

生物工学

Detection and Monitoring of Tumor Associated Circulating DNA in Patient Biofluids

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular sub-classification and temporal monitoring of tumor response to therapy. Here, we describe our dPCR-based method for the detection of tumor somatic mutations in cell free DNA (cfDNA), readily available in&#160;patient biofluids. Although limited in the number of mutations that can be tested for in each assay, this method provides a high level of sensitivity and specificity. Monitoring of mutation abundance, as calculated by MAF, allows for the evaluation of tumor response to therapy, thereby providing a much-needed supplement to radiographic imaging.

Here, we present a protocol to detect tumor somatic mutations in circulating DNA present in patient biological fluids (biofluids). Our droplet digital polymerase chain reaction (dPCR)-based method enables quantification of the tumor mutation allelic frequency (MAF), facilitating a minimally invasive complement to diagnosis and temporal monitoring of tumor response.

患者の生体流体における腫瘍関連循環DNAの検出とモニタリング

Complications associated with upfront and repeat surgical tissue sampling present the need for minimally invasive platforms capable of molecular ...

がん研究

Enrichment of Native Lipoprotein Particles with microRNA and Subsequent Determination of Their Absolute/Relative microRNA Content and Their Cellular Transfer Rate

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of noncoding microRNA (miRNA). In general, miRNA alters the protein expression profile due to interactions with messenger-RNA (mRNA). Thus, knowledge of the relative and absolute miRNA content of lipoprotein particles is essential to estimate the biological effect of cellular particle uptake. Here, a quantitative real-time polymerase chain reaction (qPCR)-based protocol is presented to determine the absolute miRNA content of lipoprotein particles&#8212;exemplified shown for native and miRNA-enriched lipoprotein particles. The relative miRNA content is quantified using multiwell microfluidic array cards. Furthermore, this protocol allows scientists to estimate the cellular miRNA and, thus, the lipoprotein particle uptake rate. A significant increase of the cellular miRNA level is observable when using high-density lipoprotein (HDL) particles artificially loaded with miRNA, whereas incubation with native HDL particles yields no significant effect due to their rather low miRNA content. In contrast, the cellular uptake of low-density lipoprotein (LDL) particles&#8212;neither with native miRNA nor artificially loaded with it&#8212;did not alter the cellular miRNA level.

Here, a quantitative real-time polymerase chain reaction-based protocol is presented for the determination of the native micro-RNA content (absolute/relative) of lipoprotein particles. In addition, a method for increasing the micro-RNA level, as well as a method for determining the cellular uptake rate of lipoprotein particles, is demonstrated.

マイクロ Rna による天然リポタンパク質粒子の濃縮とその絶対/相対マイクロ Rna 含量とその細胞移動速度の決定

Lipoprotein particles are predominately transporters of lipids and cholesterol in the bloodstream. Furthermore, they contain small amounts of strands of ...

生化学

Digital PCR for Quantifying Circulating MicroRNAs in Acute Myocardial Infarction and Cardiovascular Disease

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released from the cardiovascular cells into the circulation. Circulating miRNAs are highly stable and can be quantified. The quantitative expression of specific miRNAs can be linked to the pathology, and some miRNAs show high tissue and disease specificity. Finding novel biomarkers for cardiovascular diseases is of importance for medical research. Quite recently, digital polymerase chain reaction (dPCR) has been invented. dPCR, combined with fluorescent hydrolysis probes, enables specific direct absolute quantification. dPCR exhibits superior technical qualities, including a low variability, high linearity, and high sensitivity compared to the quantitative polymerase chain reaction (qPCR). Thus, dPCR is a more accurate and reproducible method for directly quantifying miRNAs, particularly for the use in large multi-center cardiovascular clinical trials. In this publication, we describe how to effectively perform digital PCR in order to assess the absolute copy number in serum samples.

Circulating microRNAs have shown promise as biomarkers for cardiovascular diseases and acute myocardial infarctions. In this study, we describe a protocol for miRNA extraction, reverse transcription, and digital PCR for the absolute quantification of miRNAs in the serum of patients with cardiovascular disease.

急性心筋梗塞と心血管疾患のマイクロ Rna を循環の定量化デジタル PCR

Circulating serum microRNAs (miRNAs) have shown promise as biomarkers for the cardiovascular disease and acute myocardial infarction (AMI), being released ...

医学

Highly Efficient Ligation of Small RNA Molecules for MicroRNA Quantitation by High-Throughput Sequencing

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide resolution. This technique captures miRNAs by attaching 3&#8217; and 5&#8217; oligonucleotide adapters to miRNA molecules and allows de novo miRNA discovery. Coupling with powerful next-generation sequencing platforms, miR-Seq has been instrumental in the study of miRNA biology. However, significant biases introduced by oligonucleotide ligation steps have prevented miR-Seq from being employed as an accurate quantitation tool. Previous studies demonstrate that biases in current miR-Seq methods often lead to inaccurate miRNA quantification with errors up to 1,000-fold for some miRNAs1,2. To resolve these biases imparted by RNA ligation, we have developed a small RNA ligation method that results in ligation efficiencies of over 95% for both 3&#8217; and 5&#8242; ligation steps. Benchmarking this improved library construction method using equimolar or differentially mixed synthetic miRNAs, consistently yields reads numbers with less than two-fold deviation from the expected value. Furthermore, this high-efficiency miR-Seq method permits accurate genome-wide miRNA profiling from in vivo total RNA samples2.

MicroRNAs (miRNAs) are a widely conserved class of regulatory molecules. Here we describe a miRNA cloning method that relies upon two potent ligation steps followed by high-throughput sequencing. Our method permits accurate genome-wide quantitation of miRNAs.

ハイスループットシークエンシングによりマイクロRNA定量のための低分子RNA分子の高効率ライゲーション

MiRNA cloning and high-throughput sequencing, termed miR-Seq, stands alone as a transcriptome-wide approach to quantify miRNAs with single nucleotide ...

生物学

Probe-based Real-time PCR Approaches for Quantitative Measurement of microRNAs

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that provides an accurate and reproducible analysis. Probe-based chemistry offers the least background fluorescence as compared to other (dye-based) chemistries. Presently, there are several platforms available that use probe-based chemistry to quantitate transcript abundance. qPCR in a 96 well plate is the most routinely used method, however only a maximum of 96 samples or miRNAs can be tested in a single run. This is time-consuming and tedious if a large number of samples/miRNAs are to be analyzed. High-throughput probe-based platforms such as microfluidics (e.g. TaqMan Array Card) and nanofluidics arrays (e.g. OpenArray) offer ease to reproducibly and efficiently detect the abundance of multiple microRNAs in a large number of samples in a short time. Here, we demonstrate the experimental setup and protocol for miRNA quantitation from serum or plasma-EDTA samples, using probe-based chemistry and three different platforms (96 well plate, microfluidics and nanofluidics arrays) offering increasing levels of throughput.

Circulating microRNAs have recently emerged as promising and novel biomarkers for various cancers and other diseases. The goal of this article is to discuss three different probe-based real-time PCR platforms and methods that are available to quantify and determine the abundance of circulating microRNAs.

プローブベースのリアルタイムPCRは、マイクロRNAの定量的測定のためのアプローチ

Probe-based quantitative PCR (qPCR) is a favoured method for measuring transcript abundance, since it is one of the most sensitive detection methods that ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

デジタル核酸定量アッセイの簡単な一括読み出し

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Measuring Single-Cell Mitochondrial DNA Copy Number and Heteroplasmy Using Digital Droplet Polymerase Chain Reaction

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron transport chain. Unlike the diploid nuclear genome, most cells contain many more copies of mtDNA, ranging from less than 100 to over 200,000 copies depending on cell type. MtDNA copy number is increasingly used as a biomarker for a number of age-related degenerative conditions and diseases, and thus, accurate measurement of the mtDNA copy number is becoming a key tool in both research and diagnostic settings. Mutations in the mtDNA, often occurring as single nucleotide polymorphisms (SNPs) or deletions, can either exist in all copies of the mtDNA within the cell (termed homoplasmy) or as a mixture of mutated and WT mtDNA copies (termed heteroplasmy). Heteroplasmic mtDNA mutations are a major cause of clinical mitochondrial pathology, either in rare diseases or in a growing number of common late-onset diseases such as Parkinson's disease. Determining the level of heteroplasmy present in cells is a critical step in the diagnosis of rare mitochondrial diseases and in research aimed at understanding common late-onset disorders where mitochondria may play a role. MtDNA copy number and heteroplasmy have traditionally been measured by quantitative (q)PCR-based assays or deep sequencing. However, the recent introduction of ddPCR technology has provided an alternative method for measuring both parameters. It offers several advantages over existing methods, including the ability to measure absolute mtDNA copy number and sufficient sensitivity to make accurate measurements from single cells even at low copy numbers. Presented here is a detailed protocol describing the measurement of mtDNA copy number in single cells using ddPCR, referred to as droplet generation PCR henceforth, with the option for simultaneous measurement of heteroplasmy in cells with mtDNA deletions. The possibility of expanding this method to measure heteroplasmy in cells with mtDNA SNPs is also discussed.

Here we present a protocol for measuring absolute mitochondrial (mt)DNA copy number and mtDNA deletion heteroplasmy levels in single cells.

デジタル液滴ポリメラーゼ連鎖反応を用いた単一細胞ミトコンドリアDNAコピー数とヘテロプラスミーの測定(英語)

The mammalian mitochondrial (mt)DNA is a small, circular, double-stranded, intra-mitochondrial DNA molecule, encoding 13 subunits of the electron ...

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

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 approach is appropriate for examining physiological changes in target gene expression levels. However, absolute quantification using better statistical analysis is preferable for a comprehensive assessment of gene expression levels. Absolute quantification is still not in common use. This report describes a protocol for measuring the absolute levels of plasma miRNA, using RT-qPCR with or without pre-amplification.
A fixed volume (200 &#181;L) of EDTA-plasma was prepared from the blood collected from the femoral vein of conscious cynomolgus monkeys (n = 50). Total RNA was extracted using commercially available system. Plasma miRNAs were quantified by probe-based RT-qPCR assays which contains miRNA-specific forward/reverse PCR primer and probe. Standard curves for absolute quantification were generated using commercially available synthetic RNA oligonucleotides. A synthetic cel-miR-238 was used as an external control for normalization and quality assessment. The miRNAs that showed quantification cycle (Cq) values above 35 were pre-amplified prior to the qPCR step.
Among the 8 miRNAs examined, miR-122, miR-133a, and miR-192 were detectable without pre-amplification, whereas miR-1, miR-206, and miR-499a required pre-amplification because of their low expression levels. MiR-208a and miR-208b were not detectable even after pre-amplification. Sample processing efficiency was evaluated by the Cq values of the spiked cel-miR-238. In this assay method, technical variation was estimated to be less than 3-fold and the lower limit of quantification (LLOQ) was 102 copy/&#181;L, for most of the examined miRNAs.
This protocol provides a better estimate of the quantity of plasma miRNAs, and allows quality assessment of corresponding data from different studies. Considering the low number of miRNAs in body fluids, pre-amplification is useful to enhance detection of poorly expressed miRNAs.

This report describes a protocol for measuring the absolute levels of plasma miRNA, using quantitative real-time reverse transcription PCR with or without pre-amplification. This protocol affords better understanding of the quantity of plasma miRNAs and allows qualitative assessment of corresponding data from different studies or laboratories.

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 ...

Quantification of Circular RNAs Using Digital Droplet PCR

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 water-in-oil droplets, and the PCR occurs in the individual droplets. The dd-PCR has several advantages over conventional real-time qPCR, including increased accuracy in detecting low-abundance targets, omitting reference genes for quantification, eliminating technical replicates for samples, and showing high resilience to inhibitors in the samples. Recently, dd-PCR has become one of the most popular methods for accurately quantifying target DNA or RNA for gene expression analysis and diagnostics. Circular RNAs (circRNAs) are a large family of recently discovered covalently closed RNA molecules lacking 5' and 3' ends. They have been shown to regulate gene expression by acting as sponges for RNA-binding proteins and microRNAs. Furthermore, circRNAs are secreted into body fluids, and their resistance to exonucleases makes them serve as biomarkers for disease diagnosis. This article aims to show how to perform divergent primer design, RNA extraction, cDNA synthesis, and dd-PCR analysis to accurately quantify specific circular RNA (circRNA) levels in cells. In conclusion, we demonstrate the precise quantification of circRNAs using dd-PCR.

This protocol describes the detailed method of digital droplet PCR (dd-PCR) for precise quantification of circular RNA (circRNA) levels in cells using divergent primers.

デジタル液滴PCRを用いた環状RNAの定量

Digital droplet polymerase chain reaction (dd-PCR) is one of the most sensitive quantification methods; it fractionates the reaction into nearly 20,000 ...