Name: cell-free dna integrity analysis in urine samples
Uploaded: 2017-01-05T14:00:00.000+00:00
Duration: 7 min 58 s
Description: Watch this Scientific Journal Video about Cell-Free DNA Integrity Analysis in Urine Samples at JoVE.com

The authors thank Gr&#225;inne Tierney and Silvia Bellissimo for their editorial assistance.

该协议遵循IRST人类研究伦理委员会的指导方针。 注:在这个协议中，我们提取的DNA从尿液样本以进行DNA UCF完整性分析。 LNCaP和MRC细胞系用于构建标准。技术如DNA提取，DNA定量（分光光度计和实时PCR的对照基因，STOX1），以及对特定的癌基因的实时PCR进行（ 图1）。 1.尿液收集与处理<ol><li>获得清洁，干燥，塑料杯干净的副渔获物第一晨尿样品。收集至少50毫升的尿液样本。 </li><li>保持尿液，在4℃下不超过3小时，它在相同的温度发送至实验室。 </li><li>通过在实验室和转印到达时立即两次反相它分成两个50-mL锥形底聚丙烯管中混合各样品。 </li><li>离心管在850×g离心在4℃下或在室温下10分钟。 </li><li>小心转移10毫升尿上清的上部成两个5-mL管，而使细胞沉淀上面的上清液中的至少2毫升。 注意:传输上清液的上部降低污染的被细胞或细胞碎片的风险。有上部和下部部分之间没有明确的划分。 </li><li>弃沉淀，并立即在-80冻结上清液℃直至使用。 </li></ol> 2.从尿液上清，细胞系DNA分离注:来自细胞系隔离的DNA（ 例如，LNCAP前列腺癌或MRC膀胱癌）使用商业试剂盒并按照制造商的说明。应使用商业协议来执行从尿上清液DNA的分离，修改如下: <ol><li>解冻尿上清液在室温下的一个等分试样。&lt;/ LI&gt; <li>涡和混合尿样和转移尿上清液1毫升到一个清晰的5毫升管。冷冻在-80℃的残余尿。 </li><li>添加100微升蛋白酶K的直接向样品。 </li><li>添加1毫升AL缓冲到样品并吹打混匀。 </li><li>关闭管，并在56℃孵育样品15分钟。 </li><li>孵育期间，制备每个样品一列和制备洗涤缓冲液，AW1和AW2，按照制造商的说明（加100％的乙醇的指示量）。 </li><li>使样品回到室温，并添加1毫升的无水乙醇中。吹打充分拌匀。 </li><li>添加650微升在步骤2.7到塔中获得的混合物中，并在6000 xg离心离心它为1分钟。 </li><li>丢弃含有通流管，并放置在塔上的新的，干净的收集管。直到所有的样品混合物的已使用的（5次）重复步骤2.8和2.9。 </li><li>添加500＃181; l缓冲AW1无润湿柱的边缘。离心它在6000 xg离心1分钟。 </li><li>丢弃该管含有流通并用新的，干净的收集管更换。 </li><li>添加缓冲液AW2的500微升无润湿塔的边缘。全速（20,000 XG）3 min离心它。 </li><li>丢弃该管含有流通并用新的，干净的收集管更换。 </li><li>重复步骤2.12以除去任何残留的洗涤缓冲液。 </li><li>将列到一个干净的1.5毫升管。添加洗脱缓冲液AE中的50微升并等待7分钟，以确保缓冲区润湿列。 </li><li>离心它在8000 xg离心1分钟。 </li><li>吸管从步骤2.15洗脱液进入微型柱中，在最大速度离心它为1分钟，以确保DNA的最大回收。 </li></ol> 3. DNA定量和稀释<ol><li>使用分光光度计，从机器人进行DNA的定量小时的细胞系和尿上清液样品。使用2μL的样品在台式光谱仪，按照制造商的说明。 </li><li>稀释UCF DNA样品以获得1毫微克/微升的浓度，并于-20℃下将DNA储存，直到DNA的完整性分析。 注意:如果该DNA量不足以与实时PCR（至少100纳克）来进行，执行新的DNA分离过程。 </li><li>稀从细胞系样品的DNA，以获得六类标准不同浓度:0.001，0.01,0.1，1,0.5，和2纳克/微升，每100微升的体积（ST1，ST2，ST3，ST4，ST5，并ST6）。在-20℃直到DNA完整性分析存储细胞系DNA的标准。 </li></ol> 4. DNA完整性检测 - PCR <ol><li>解冻的引物（浓度取决于测定的类型），绿色超混合液，细胞系的DNA的标准，并在冰上UCF DNA稀释的样品。 </li><li>准备带管的托盘f或72孔转子盘。等分试样10μL的一式两份每个标准和稀释试样和无RNase水10μL的阴性对照。 </li><li>制备的每种引物的1微升的组合（浓度如表1所示），Green超12.5微升，和6.5微升的无RNase水各样品。当准备搭配，请使用以下的样本数量:6标准的2次重复，样品数量为2的重复，2重复阴性对照，以及2个额外的样本。 </li><li>分装15μL混合到每口井的。不要吸管或旋管。 </li><li>开始与在表1所示的PCR条件的协议。 注:29×2样品+ 6×2标准+阴性对照×2 = 72（UCF脱氧核糖核酸值）:对于在UCF脱氧核糖核酸值，29 DNA样品使用72孔转子盘中的每个实时实验处理。 注:为UCF DNA完整性分析该协议还可以使用另一个PCR实时仪（使用其他设备时，ROX染料可能需要添加）进行。 </li></ol> 5. DNA完整性检测 - 数据分析与解读注:各样品的UCF DNA值是通过使用标准曲线构造为每个单独的PCR基因评价，并使用标准曲线内插实时仪器检测系统软件获得，如先前所述7-9（ 图2）。 <ol><li>评估重复;样本复制与差≥1的Ct必须被丢弃，并在第二个实验中重新评估。 </li><li>计算位数的Ct每个样品和Ct值≤36考虑样本。 </li><li>评估使用熔点分析的PCR产物的特异性。 </li><li>通过评估与标准曲线插补每个扩增子的浓度;获得每个扩增子的浓度值（纳克/μL）。</li>
</ol>

<ol>
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The authors declare no competing financial interests.

UCF DNA完整性分析是在尿评估DNA完整性一种新的，非侵入性的方法。它最近提出了膀胱9和前列腺癌7,8的早期诊断。有许多优点和UCF DNA完整性检测的缺点在这里讨论，未来的前景一起。 该方法的主要优点是，它提供了一种廉价的，非侵入性的方法和简单的协议来研究尿作为生物标志物的一个潜在来源，只需要的分子生物学技术的基本知识。该测试是快速执行，...

无细胞的DNA可以在血液和尿液由于细胞死亡通过细胞凋亡或坏死的机制来检测。血液中的游离细胞DNA已被广泛地研究了在各种疾病的诊断和预后目的，尤其是癌症1。然而，较少有人知道尿无细胞（UCF）DNA的作用。 UCF DNA可以从血液穿过肾小球滤过系统或从直接来与该体液2（ 例如，上皮细胞或前列腺细胞）接触的细胞产生。作为生物标志物的来源，使用的UCF的DNA已经主要研究了肾，膀胱癌，和前列腺癌的早期诊断由于UCF的DNA直接从尿道细胞3,4-未来的高百分比。 鲜为人知的是，UCF DNA以及分离和鉴定的最佳方法。给定的假设肿瘤细胞释放比正常细胞较长的DNA片段，评价无细胞DNA完整性已被研究以试图阐明DNA的原点在血液循环中5。一些研究表明，在血液中的游离细胞DNA完整性表示用于多种癌症6良好的诊断测试，并且在同一假设已经关于拟尿7-9。 本文介绍了一个潜在的应用膀胱和前列腺癌检测DNA UCF完整性分析的新方法。特别是，UCF DNA的完整性片段长度大于250 bp的已知的4个区域测试，以在实体肿瘤，包括前列腺癌和膀胱癌增加的DNA拷贝数:C-MYC（8q24.21），HER2（17q12.1 ），BCAS1（20q13.2）和AR（Xq12）10-14。特定癌基因，而不是随机的序列，被选择来增加癌症患者的无细胞级分发现它们的概率。之一的主要优点这种方法是，它是柔性的，并且其他区域也可以被肿瘤的类型和特性的基础上选择的。

<table><tbody><tr><td>QIAamp DNA Mini Kit&lt;br/&gt; &amp;nbsp;</td><td>Qiagen</td><td>51304</td><td></td></tr><tr><td>iQ SYBR&amp;nbsp;Green Supermix, 100 x 50 &amp;micro;L rxns, 2.5 mL (2 x 1.25 mL)</td><td>Biorad</td><td>1708880</td><td></td></tr><tr><td>IDT custom DNA oligos&amp;nbsp;</td><td>IDT&amp;nbsp;</td><td></td><td>HPLC purification, 100nMole DNA oligo</td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>Thermo Scientific</td><td></td><td>Other spectrophotometric methods could also be used to quantify DNA</td></tr><tr><td>Rotor-Gene 6000</td><td>Corbett</td><td></td><td>Another Real Time PCR instrument could also be used</td></tr><tr><td>microcentrifuge</td><td></td><td></td><td></td></tr><tr><td>one centrifuge for 50 mL tubes</td><td></td><td></td><td></td></tr><tr><td>incubator</td><td></td><td></td><td></td></tr><tr><td>-80 &amp;deg;C freezer</td><td></td><td></td><td></td></tr><tr><td>-20 &amp;deg;C freezer</td><td></td><td></td><td></td></tr><tr><td>10 &amp;#956;L pipette</td><td></td><td></td><td></td></tr><tr><td>20 &amp;#956;L pipette</td><td></td><td></td><td></td></tr><tr><td>200 &amp;#956;L pipette</td><td></td><td></td><td></td></tr><tr><td>1,000 &amp;#956;L pipette</td><td></td><td></td><td></td></tr><tr><td>pipette tips (10; 20; 200; 1,000)</td><td></td><td></td><td></td></tr><tr><td>1.5 mL tubes</td><td></td><td></td><td></td></tr><tr><td>50 mL tubes</td><td></td><td></td><td></td></tr><tr><td>15 mL tubes</td><td></td><td></td><td></td></tr><tr><td>Rotor-Disc 72 Rotor</td><td>Corbett</td><td>9018899</td><td></td></tr><tr><td>Strip Tubes and Caps, 0.1 mL (250)</td><td>Qiagen</td><td>981103</td><td></td></tr><tr><td>Collection Tubes (2 mL)</td><td>Qiagen</td><td>19201</td><td></td></tr><tr><td>Buffer AL (264 mL)</td><td>Qiagen</td><td>19075</td><td></td></tr><tr><td>Proteinase K (10 mL)</td><td>Qiagen</td><td>19133</td><td></td></tr></tbody></table>

cell-free dna integrity analysis in urine samples

尽管在血浆或血清中的循环无细胞DNA的存在已被广泛证明是生物标记物的合适来源为许多类型的癌症，很少研究都集中在可能利用尿无细胞（UCF）的DNA。从正常凋亡细胞产生高度片段化DNA的假设和癌细胞开始释放更长的DNA，评价为能够患者前列腺或膀胱癌和健康个体之间进行区分的早期诊断标志物的UCF DNA完整性的潜在作用。 一个UCF DNA完整性分析，提出的超过250 bp的长四个序列四个实时定量PCR的基础上:C-MYC，BCAS1，HER2和AR。经常有在膀胱和前列腺癌的增加的DNA拷贝数序列被选择用于分析，但该方法是灵活的，这些基因可以与INTE的其他基因被取代休息。 UCF的DNA作为生物标志物的来源的潜在效用已被证实为泌尿系统恶性肿瘤，从而为在UCF DNA鉴定进一步研究的方式。在UCF DNA完整性测试具有作为非侵入性，快速，容易执行，以进行分析所需的尿只有几毫升的优点。

总游离DNA浓度是通过分光光度法量化的所有样品进行分析，呈现出范围1.51和138之间纳克/微升。五个控制样品用于数据的再现性:两个独立的实时实验对c-MYC，HER2，BCAS1，AR和STOX1进行。变化（CV）的系数，然后计算每个基因（ 表2），用两个独立的实验之间的良好度再现性（ 表2）。 <p class="jove_content" fo:keep...

Watch this Scientific Journal Video about Cell-Free DNA Integrity Analysis in Urine Samples at JoVE.com

Cell-Free DNA Integrity Analysis in Urine Samples

A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

无细胞DNA完整性分析尿样中

Although the presence of circulating cell-free DNA in plasma or serum has been widely shown to be a suitable source of biomarkers for many types of ...

cell-free-dna-integrity-analysis-in-urine-samples

Universidad de Cartagena UDEC

Research

JoVE Journal

Cancer Research

13.7K 次观看.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. A method for analyzing DNA integrity in the cell-free supernatant fraction of urine samples is proposed. The method is suitable for early detection of urological malignancies and has proven accurate for the early diagnosis of bladder cancer.

视频: Cell-Free DNA Integrity Analysis in Urine Samples

Single Droplet Digital Polymerase Chain Reaction for Comprehensive and Simultaneous Detection of Mutations in Hotspot Regions

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation into thousands of nano-sized water-in-oil individual reactions. Recently, ddPCR has become one of the most accurate and sensitive tools for circulating tumor DNA (ctDNA) detection. One of the major limitations of the standard ddPCR technique is the restricted number of mutations that can be screened per reaction, as specific hydrolysis probes recognizing each possible allelic version are required. An alternative methodology, the drop-off ddPCR, increases throughput, since it requires only a single pair of probes to detect and quantify potentially all genetic alterations in the targeted region. Drop-off ddPCR displays comparable sensitivity to conventional ddPCR assays with the advantage of detecting a greater number of mutations in a single reaction. It is cost-effective, conserves precious sample material, and can also be used as a discovery tool when mutations are not known a priori.

Here, we present a protocol to accurately quantify multiple genetic alterations of a target region in a single reaction using drop-off ddPCR and a unique pair of hydrolysis probes.

单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

Droplet digital polymerase chain reaction (ddPCR) is a highly sensitive quantitative polymerase chain reaction (PCR) method based on sample fractionation ...

科研

遗传学

Evaluation of Colorectal Cancer Risk and Prevalence by Stool DNA Integrity Detection

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a reliable probability of the presence of pre-neoplastic or neoplastic colorectal lesions. This method, called fluorescence long DNA (FL-DNA), is a fast, non-invasive procedure that is an improvement upon the primary prevention system. This method is based on evaluation of fecal DNA integrity by quantitative amplification of specific targets of genomic DNA. In particular, the evaluation of DNA fragments longer than 200 bp allows for detection of patients with colorectal lesions with very high specificity. However, this system and all currently available stool DNA tests present some general issues that need to be addressed (e.g., the frequency at which tests should be carried out and optimal number of stool samples collected at each timepoint for each individual). However, the main advantage of FL-DNA is the possibility to use it in association with a test currently used in the CRC screening program, known as the immunochemical-based fecal occult blood test (iFOBT). Indeed, both tests can be performed on the same sample, reducing costs and achieving a better prediction of the eventual presence of colorectal lesions.

The presented diagnostic FL-DNA kit is a time-saving and user-friendly method to determine the reliable probability of the presence of colorectal cancer lesions.

通过大刀DNA完整性检测评估结肠直肠癌风险和患病率

Nowadays, stool DNA can be isolated and analyzed by several methods. The long fragments of DNA in stool can be detected by a qPCR assay, which provides a ...

癌症研究

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.

患者生物流体中肿瘤相关循环DNA的检测与监测

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

Detection of Cell-Free DNA in Blood Plasma Samples of Cancer Patients

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis as well as for the treatment selection and its response in cancer patients. The current gold standard method for detecting tumor mutations involves a genetic test of tumor DNA by means of tumor biopsies. However, this invasive method is difficult to be performed repeatedly as a follow-up test of the tumor mutational repertoire. Liquid biopsy is a new and emerging technique for detecting tumor mutations as an easy-to-use and non-invasive biopsy approach.
Cancer cells multiply rapidly. In parallel, numerous cancer cells undergo apoptosis. Debris from these cells are released into a patient&#8217;s circulatory system, together with finely fragmented DNA pieces, called cell-free DNA (cfDNA) fragments, which carry tumor DNA mutations. Therefore, for identifying cfDNA based biomarkers using liquid biopsy technique, blood samples are collected from the cancer patients, followed by the separation of plasma and buffy coat. Next, plasma is processed for the isolation of cfDNA, and the respective buffy coat is processed for the isolation of a patient&#39;s genomic DNA. Both nucleic acid samples are then checked for their quantity and quality; and analyzed for mutations using next-generation sequencing (NGS) techniques.
In this manuscript, we present a detailed protocol for liquid biopsy, including blood collection, plasma, and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

In this paper we present a detailed protocol for non-invasive liquid biopsy technique, including blood collection, plasma and buffy coat separation, cfDNA and germline DNA extraction, quantification of cfDNA or germline DNA, and cfDNA fragment enrichment analysis.

癌症患者血浆样本中无细胞DNA检测

Identifying mutations in tumors of cancer patients is a very important step in disease management. These mutations serve as biomarkers for tumor diagnosis ...

A Standardized Liquid Biopsy Preanalytical Protocol for Downstream Circulating-Free DNA Applications

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that originate from the primary and/or metastatic tumor. LB has emerged as a mainstay in translational research and has started to become part of clinical oncology practice, providing a minimally invasive alternative to solid biopsy. The LB allows real-time monitoring of a tumor via a minimally invasive sample extraction, such as blood. The applications include early cancer detection, patient follow-up for the detection of disease progression, assessment of minimal residual disease, and potential identification of molecular progression and mechanism of resistance. In order to achieve a reliable analysis of these samples that can be reported in the clinic, the preanalytical procedures should be carefully considered and strictly followed. Sample collection, quality, and storage are crucial steps that determine their usefulness in downstream applications. Here, we present standardized protocols from our liquid biopsy working module for collecting, processing, and storing plasma and serum samples for downstream liquid biopsy analysis based on circulating-free DNA. The protocols presented here require standard equipment and are sufficiently flexible to be applied in most laboratories focused on biological procedures.

The liquid biopsy has revolutionized our approach to oncology translational studies, with sample collection, quality, and storage being crucial steps for its successful clinical application. Here we describe a standardized and validated protocol for downstream circulating-free DNA applications that can be applied in most translational research laboratories.

用于下游无循环DNA应用的标准化液体活检前分析方案

The term liquid biopsy (LB) refers to molecules such as proteins, DNA, RNA, cells, or extracellular vesicles in blood and other bodily fluids that ...

Uracil-DNA Glycosylase Assay by Matrix-assisted Laser Desorption/Ionization Time-of-flight Mass Spectrometry Analysis

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of cytosine. Thus, it is crucial for genome integrity maintenance. A highly specific, non-labeled, non-radio-isotopic method was developed to measure UDG activity. A synthetic DNA duplex containing a site-specific uracil was cleaved by UDG and then subjected to Matrix-assisted Laser Desorption/Ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis. A protocol was established to preserve the apurinic/apyrimidinic site (AP) product in DNA without strand break. The change in the m/z value from the substrate to the product was used to evaluate uracil hydrolysis by UDG. A G:U substrate was used for UDG kinetic analysis yielding the Km = 50 nM, Vmax = 0.98 nM/s, and Kcat = 9.31 s-1. Application of this method to a uracil glycosylase inhibitor (UGI) assay yielded an IC50 value of 7.6 pM. The UDG specificity using uracil at various positions within single-stranded and double-stranded DNA substrates demonstrated different cleavage efficiencies. Thus, this simple, rapid, and versatile MALDI-TOF MS method could be an excellent reference method for various monofunctional DNA glycosylases. It also has the potential as a tool for DNA glycosylase inhibitor screening.

A non-labeled, non-radio-isotopic method to assay uracil-DNA glycosylase activity was developed using MALDI-TOF mass spectrometry for direct apurinic/apyrimidinic site-containing product analysis. The assay proved to be quite simple, specific, rapid, and easy to use for DNA glycosylase measurement.

尿嘧啶-DNA糖基化酶测定，采用基质辅助激光解吸/电离飞行时间质谱分析

Uracil-DNA glycosylase (UDG) is a key component in the base excision repair pathway for the correction of uracil formed from hydrolytic deamination of ...

生物化学

Quantification of three DNA Lesions by Mass Spectrometry and Assessment of Their Levels in Tissues of Mice Exposed to Ambient Fine Particulate Matter

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are electrophilic, generate reactive electrophiles upon biotransformation, or induce oxidative stress. Among the oxidized nucleobases, the most studied one is 8-oxo-7,8-dihydroguanine (8-oxoGua) or 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), a biomarker of oxidatively induced base damage in DNA. Aldehydes and epoxyaldehydes resulting from the lipid peroxidation process are electrophilic molecules able to form mutagenic exocyclic DNA adducts, such as the etheno adducts 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-&#949;dGuo) and 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-&#949;dAdo), which have been suggested as potential biomarkers in the pathophysiology of inflammation. Selective and sensitive methods for their quantification in DNA are necessary for the development of preventive strategies to slow down cell mutation rates and chronic disease development (e.g., cancer, neurodegenerative diseases). Among the sensitive methods available for their detection (high performance liquid chromatography coupled to electrochemical or tandem mass spectrometry detectors, comet assay, immunoassays, 32P-postlabeling), the most selective are those based on high performance liquid chromatography coupled to tandem mass spectrometry (HPLC-ESI-MS/MS). Selectivity is an essential advantage when analyzing complex biological samples and HPLC-ESI-MS/MS evolved as the gold standard for quantification of modified nucleosides in biological matrices, such as DNA, urine, plasma and saliva. The use of isotopically labeled internal standards adds the advantage of corrections for molecule losses during the DNA hydrolysis and analyte enrichment steps, as well as for differences of the analyte ionization between samples. It also aids in the identification of the correct chromatographic peak when more than one peak is present.
We present here validated sensitive, accurate and precise HPLC-ESI-MS/MS methods that were successfully applied for the quantification of 8-oxodGuo, 1,N6-dAdo and 1,N2-dGuo in lung, liver and kidney DNA of A/J mice for the assessment of the effects of ambient PM2.5 exposure.

We describe here methods for sensitive and accurate quantification of the lesions 8-oxo-7,8-dihydro-2&#39;-deoxyguanosine (8-oxodGuo), 1,N6-etheno-2&#39;-deoxyadenosine (1,N6-dAdo) and 1,N2-etheno-2&#39;-deoxyguanosine (1,N2-dGuo) in DNA. The methods were applied to the assessment of the effects of ambient fine particulate matter (PM2.5) in tissues (lung, liver and kidney) of exposed A/J mice.

环境细颗粒物小鼠组织中三种 DNA 损伤的质谱定量及水平评价

DNA adducts and oxidized DNA bases are examples of DNA lesions that are useful biomarkers for the toxicity assessment of substances that are ...

免疫与感染

用于癌症诊断的 CRISPR-Cas 合成尿液生物标志物测试

CRISPR-Cas-mediated Multianalyte Synthetic Urine Biomarker Test for Portable Diagnostics

Creating synthetic biomarkers for the development of precision diagnostics has enabled detection of disease through pathways beyond those used for traditional biofluid measurements. Synthetic biomarkers generally make use of reporters that provide readable signals in the biofluid to reflect the biochemical alterations in the local disease microenvironment during disease incidence and progression. The pharmacokinetic concentration of the reporters and biochemical amplification of the disease signal are paramount to achieving high sensitivity and specificity in a diagnostic test. Here, a cancer diagnostic platform is built using one format of synthetic biomarkers: activity-based nanosensors carrying chemically stabilized DNA reporters that can be liberated by aberrant proteolytic signatures in the tumor microenvironment. Synthetic DNA as a disease reporter affords multiplexing capability through its use as a barcode, allowing for the readout of multiple proteolytic signatures at once. DNA reporters released into the urine are detected using CRISPR nucleases via hybridization with CRISPR RNAs, which in turn produce a fluorescent or colorimetric signal upon enzyme activation. In this protocol, DNA-barcoded, activity-based nanosensors are constructed and their application is exemplified in a preclinical mouse model of metastatic colorectal cancer. This system is highly modifiable according to disease biology and generates multiple disease signals simultaneously, affording a comprehensive understanding of the disease characteristics through a minimally invasive process requiring only nanosensor administration, urine collection, and a paper test which enables point-of-care diagnostics.

作者聚焦:用于疾病检测和成像的工程分子工具

This protocol describes a CRISPR-Cas-mediated, multianalyte synthetic urine biomarker test that enables point-of-care cancer diagnostics through the ex vivo analysis of tumor-associated protease activities.

无细胞DNA完整性分析尿样中

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单液滴数字聚合酶链反应对热点地区突变的综合和同步检测

通过大刀DNA完整性检测评估结肠直肠癌风险和患病率

患者生物流体中肿瘤相关循环DNA的检测与监测

癌症患者血浆样本中无细胞DNA检测

用于下游无循环DNA应用的标准化液体活检前分析方案

尿嘧啶-DNA糖基化酶测定，采用基质辅助激光解吸/电离飞行时间质谱分析

环境细颗粒物小鼠组织中三种 DNA 损伤的质谱定量及水平评价

CRISPR-Cas介导的多分析物合成尿液生物标志物检测用于便携式诊断

CRISPR-Cas介导的多分析物合成尿液生物标志物检测用于便携式诊断

CRISPR-Cas介导的多分析物合成尿液生物标志物检测用于便携式诊断