(-, 2010/15 9980 5 mille n başına. Özel Program Moleküler Klinik Onkoloji) ve Üniversite ve Öğretim İtalyan Bakanlığı MF (MFAG 11676) Kanser Araştırma İtalyan Derneği (AIRC) den ve MN için fon tarafından desteklenen MN Araştırma FIRB 2011 (Proje RBAPIIBYNP).

Plazma ya da serum gelen mikroRNA İzolasyon Not: Plazma ve serum hazırlama miRNA miktarının dolaşımdaki ilgili bir adımdır. plazma ve serum hazırlanması için herhangi bir tercih işlemi yoktur. dikkate Önemli olan tek şey aynı deneyden tüm örnekler aynı iş akışı kullanarak işlenmelidir olmasıdır. 200 ul serum veya plazmadan başlayın. Toplam RNA ticari olarak temin edilebilir kitler kullanılarak serum veya plazma izole edilebilir. 1. (miRNA dahil) Toplam RNA Protokolü İzolasyon <ol><li> Buz üzerinde Çözülme serum / plazma örnekleri. </li><li> 200 ul serum / plazma liziz reaktifi (örn., QIAzol) 1 ml ilave edilir. </li><li> vorteks karışım, 5 dakika boyunca oda sıcaklığında tezgah üzerinde Homojenat içeren tüp yerleştirin. </li><li> C. sentetik miRNA cel-miR-39-3p bir 4.16 nM çözeltisi 3 ul ekleyin elegans. </li><li> kloroform 200 ul ekle. 15 saniye boyunca kuvvetlice tüpü çalkalayın. Yeri inci2 dakika boyunca oda sıcaklığında tezgah e tüpü. </li><li> 4 ° C&#39;de 12,000 x g&#39;de 15 dakika boyunca santrifüj aşamaları ayırma elde etmek üzere, üst sulu faz RNA içerir. </li><li> , Yeni bir tüpe 700 ul sulu faz transfer herhangi bir beyaz interfaz malzeme transferini engellemek. </li><li> % 100 etanol 1 ml ilave edilir ve çevirerek karıştırın. </li><li> Pipet yukarı mini spin kolon içine numune 700 ul 2 ml toplama tüpü yerleştirilir. 15 saniye boyunca 12.000 xg&#39;de kapağı ve santrifüj kapatın. flow-through atın. </li><li> Kalan örnek kullanarak bu adımı yineleyin. </li><li> Mini döndürme kolonuna 700 ul Tampon RWT ekle sütunu yıkamak için 12,000 xg&#39;de 15 sn için kapağı ve santrifüj kapatın. flow-through atın. </li><li> Mini spin kolon içine Pipetleyin 500 ul Tampon RPE. 12.000 rpm&#39;de 15 saniye boyunca kapağı ve santrifüj kapatın. flow-through atın. Yine bu adımı yineleyin. </li><li> spin kolon kurumasını 2 dakika süreyle tam hızda mini spin kolon santrifüjKalıntı etanolden membran. </li><li> Sütunun membran üzerinde yeni bir 1.5 ml&#39;lik bir toplama tüpü ve damlalıklı 35 mL RNaz içermeyen su mini spin kolon aktarın. </li><li> RNA elüte etmek için 12,000 x g&#39;de 1 dakika boyunca santrifüj. </li><li> -80 ° C&#39;de RNA saklayın. Not: RNA konsantrasyonu, doğru olarak belirlenebilir edemediğinden, giriş miktarı için bir ölçü olarak, sabit hacimli kullanımı. </li></ol> 2. mikroRNA Ters Transkripsiyon <ol><li> 5x reaksiyon tamponu, nükleaz içermeyen su: ters transkripsiyon (RT) reaktif karışımı bileşenleri çözülme. Yavaşça karıştırın buz üzerinde tüpler ve yeri tersini. Kullanımdan hemen önce, dondurucudan enzim karışımı çıkarın ve buz üzerine yerleştirin. tüm reaktifler aşağı doğru döndürün. </li><li> Tablo 1 &#39;de tarif edildiği gibi buz üzerinde RT reaktif karışımı hazırlayın. En az% 10 aşan karışımı hazırlayın. </li><li> Pipetleme tarafından reaksiyonu karıştırın ve daha sonra aşağı doğru döndürün. </li><li> 42 ° C&#39;de 60 dakika boyunca inkübe edin. </li><li> Rever Isı inaktive95 ° C&#39;de 5 dakika boyunca SE transkriptaz. </li><li> Hemen ardından 4 ° C&#39;ye soğutun. </li><li> -20 ° C&#39;de cDNA saklayın. </li></ol> 3. cDNA Sulandırma <ol><li> nükleaz içermeyen su içinde planlanan ddPCR reaksiyonları için gerekli olan cDNA şablon miktarının seyreltilir. 1:50 ve 1 arasında cDNA reaksiyonu sulandırmak: 500 suda, hedef miRNA bereket bağlıdır. -20 ° C&#39;de seyreltilmiş cDNA saklayın. seyreltilmiş cDNA -20 ° C&#39;de en az 3 ay boyunca stabil olduğu kanıtlanmıştır. </li></ol> 4. damlacık oluşturma ve PCR Not: damlacık oluşturma her seferinde 8 numuneler üzerinde yapılmalıdır. Teknik çoğaltır nedeniyle 12,15 .A Şablonsuz kontrol (NTC) bir numunesi, her plaka çalıştırılmalıdır bu teknolojinin yüksek ölçüde tekrarlanabilirliği ve her biri farklı ddPCR durum için gerekli değildir. <ol><li> Çözülme ve ana karışımı dengeye (örn., EvaGreen Master Mix) oda sıcaklığında, mikroRNA primer seti ve cDNA. </li><li> Master Mix th Mixoroughly Tüpü birkaç kez baş aşağı çevirerek. tüm reaktifleri aşağı doğru döndürün. </li><li> Tablo 2&#39;de tarif edildiği gibi ddPCR karışımı hazırlayın. Birden ddPCR reaksiyonlar gerçekleşirken zaman, ddPCR çalışma-çözeltisi karışımı hazırlayın. en az% 10 aşan karışımı hazırlayın. </li><li> monte kez iyice karıştırın ve ddPCR karışımı aşağı doğru döndürün. </li><li> ddPCR 12 ul, 96 oyuklu bir plaka, PCR ya da PCR tüpleri içine karıştırmak dağıtın. </li><li> de / her bir tüpe seyreltildi cDNA şablonunun 8 ul ekle. </li><li> tutucu içine damlacık jeneratör kartuşu takın. </li><li> Aktarım hava kaçınarak kuyunun dibinde kabarcıkları dikkatle ise damlacık jeneratör kartuşunun örnek kuyu (orta sıra) her Hazırlanan numune 20 ul. </li><li> damlacık jeneratör yağı 70 ul her petrol kuyusu (alt sıra) doldurun. </li><li> kartuş yuvasına üzerinde conta kanca ve damlacık jeneratör yerleştirin. </li><li> Üreticinin pr göre damlacık nesil başlatmak için kapağını kapatınotocol. damlacık oluşturma bittiğinde, kapağı açın atılabilir conta çıkarın. kartuşun üst kuyular damlacıkları içerir. </li><li> 96 oyuklu bir PCR plaka tek bir sütuna sekiz en oyuklara (damlacıklar) içeriğinin pipetleyin yavaş ve düzgün 40 ul. </li><li> hemen buharlaşmasını önlemek için damlacıkların aktardıktan sonra folyo ile PCR plaka mühür. Damlacık okuyucu iğne ile uyumlu delinebilir folyo plaka contaları kullanın. </li><li> bir plak kapatıldı, 30 dk içinde termal çevrimi (PCR) başlayın. </li><li> Tablo 3&#39;e göre bisiklet protokolü gerçekleştirin. </li></ol> 5. Damlacık Okuma <ol><li> damlacık okuyucu açın. </li><li> damlacık okuyucuya termal döngü plaka taşıyın. </li><li> plaka sahibinin tabanına PCR sonrası damlacıkları içeren 96-PCR plaka koyun. </li><li> PCR plaka üzerinde plaka sahibinin üst yerleştirin. Sıkıca hem serbest bırakma tırnaklarını basınaşağı tutucuya PCR plakasını sabitlemek için. </li><li> Sistem PC&#39;den yazılımını başlatın. <ol><li> deneyi (Mutlak miktar) tanımlamak için Kur&#39;u tıklatın ve sonra damlacık okumaya başlamak için Çalıştır &#39;ı tıklatın. </li><li> damlacık okuma tamamlandığında, tıklayın açın ve verileri analiz etmek için, lütfen &quot;Analiz&quot;. </li><li> Pozitif damlacıkları (kement aracı) (Şekil 1B) seçmek için analiz yazılımı 2D genlik arsa kullanın. </li><li> Pozitif ve toplam damlaların sayısını kontrol etmek Olaylar sekmesini kullanın. 18.000 toplam sayısı - 21.000 damlacıkları genellikle probeless ddPCR ile elde edilir. </li><li> Pozitif damlacıklar seçildikten sonra, Konsantrasyon sekmesinden ihracat .csv seçeneğini kullanarak miRNA konsantrasyon değerleri ihracat. </li></ol></li></ol>

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The authors have nothing to disclose.

Sirkülasyon miRNA&#39;lar son derece düşük konsantrasyonlarda, kanda bulunan plazma ve serum örneklerinden elde edilebilir RNA miktarı düşüktür. Bu nedenle, bu tür mikrodizi ve RNA dizi analizi gibi diğer tekniklerle ölçmek zordur. Ayrıca, veriler normalleştirme üzerinde anlaşma genel bir eksikliği ve kan endojen &quot;referans&quot; miRNA&#39;ların varlığı yoktur. Bu bağlamda, serum veya plazma çok düşük dahi olsa ml başına miRNA kopya sayısını sayma yeteneğine sahip damlacık dijital ...

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

mikroRNA (miRNA&#39;ların) (hücre içermeyen) dolaşımdaki kan akışı içine hücrelerden salınırlar. dolaşımdaki Belirli mikroRNA miktarı bir hastalık durumu ile bağlantılı hastalık belirteç olarak kullanılmak üzere bir potansiyele sahip olmuştur. Dijital PCR teknolojisi bir boya temelli kimya kullanılarak ve damlacık microRNA ölçümü dolaşan için hassas ve doğru bir yöntem son zamanlarda geliştirilmiştir. Spesifik olarak, nükleik asit (LNA) belirli miRNA&#39;ların mutlak miktarının elde edilmesi mümkün olmaktadır, uyumlu bir damlacık dijital PCR sisteminin yeşil floresan DNA bağlayıcı boya ile miRNA spesifik primerler tabanlı Kilitli kullanılarak. Burada, bu tekniği gerçekleştirmek gibi plazma ve serum gibi biyolojik sıvılarda, içinde miRNA miktarını değerlendirmek için nasıl tarif uygulanabilir ve etkili hem de.

Plazma ya da serum ml başına özgül miRNA&#39;ların mutlak miktarı, dijital PCR teknolojisi yeşil fluoresan DNA bağlayıcı boya kullanılarak ve damlacık belirlenebilir. 1 son MiRNA konsantrasyonunu belirler pozitif damlacıkları seçim sürecini sunulur şekil (kopya / ml ) analiz yazılımı ile hesaplanmıştır amplifikasyon reaksiyonunda. Kandaki her miRNA miktarı diğerlerine göre biraz miRNA türü daha bol olması, çok farklıdır. d...

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.

MicroRNA Niceleme DNA bağlayıcı Boya Kimya ve Damlacık Dijital PCR kullanarak Sirkülasyon

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

Research

JoVE Journal

Bioengineering

8.6K Görüntüleme.  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.

Video: 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.

Yüksek Verimli MicroRNA Profil: Optimize edilmiş, Multiplex QRT-PCR Fluidigm Dinamik ArrayTM IFCS Nanoliter Ölçeğinde

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

Biyomühendislik

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.

Hasta Biofluidinde tümör Ilişkili sirkülasyon DNA 'Sı tespiti ve Izlenmesi

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

Kanser Araştırmaları

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.

MicroRNA ile yerli lipoprotein parçacıkları zenginleştirme ve onların mutlak/bağıl microRNA Içerik ve onların hücresel transfer hızı sonraki belirlenmesi

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

Biyokimya

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.

Akut miyokard infarktüsü ve kardiyovasküler hastalık mikroRNA'lar dolaşan miktarının için dijital PCR

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

Tıp

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.

Yüksek Verim Dizilimine tarafından MicroRNA ölçümü için Küçük RNA molekülleri, yüksek Verimli ligasyonu

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

Biyoloji

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.

Probe-tabanlı Real-time PCR microRNA Kantitatif Ölçüm Yaklaşımları

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.

Dijital Nükleik Asit Kantifikasyon Tahliller Basit Toplu Saati

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.

Dijital Damlacık Polimeraz Zincir Reaksiyonu Kullanarak Tek Hücreli Mitokondriyal DNA Kopya Sayısını ve Heteroplazmiyi Ölçme

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.

Cynomolgus maymunlar, nicel gerçek zamanlı ters transkripsiyon PCR kullanarak plazma mikroRNA düzeylerinin mutlak miktar

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.

Dairesel RNA'ların Dijital Damlacık PCR Kullanılarak Nicelleştirilmesi

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