我们感谢 Chiara 莫利纳利和因扎吉 Martignano 在数据分析方面的支持。

该协议包括从血清或血浆样品中分离出 DNA, 以进行 real-time PCR 进行拷贝数分析。对特定靶点进行 dna 提取、dna 量控制 (分光光度计) 和 real-time PCR。在图 1中, 报告了过程和时间线的摘要。
该议定书遵循红外人类研究伦理委员会的准则。
1. 血清采集与处理
<ol>
	<li>在没有抗凝血的血清管中收集大约5毫升全血, 以获得血清。在4° c 时保持血液管直到加工。</li>
	<li>离心管在 1000 x g 为 15 min 在4° c。</li>
	<li>小心地将血清转化为2毫升的管子。</li>
	<li>丢弃收集管, 并立即冻结上清在-80 ° c, 直到使用。</li>
</ol>
2. 等离子采集与处理
<ol>
	<li>在等离子体管中收集大约10毫升全血, 用 EDTA 获得血浆。完全填充管, 然后倒置8次。在4° c 时保持血液管直到加工。</li>
	<li>离心管在 1800 x g 为15分钟在室温下没有刹车设置在离心机。</li>
	<li>小心地将血浆的上部转换成2毫升的管子。重复转移, 在白细胞层上方至少留下1毫升的上清液。 注: 上部上清的转移降低了细胞或细胞碎片的污染风险。</li>
	<li>丢弃收集管, 并立即冻结上清在-80 ° c, 直到使用。</li>
</ol>
3. 从血清或血浆中分离 DNA
注意: 从血清或血浆中分离出的 DNA 应使用以下步骤中修改的商业协议进行。
<ol>
	<li>在室温下解冻一分 (含0.5 至2毫升) 的血清或血浆。</li>
	<li>涡流和混合的样本和转移0.5 毫升的血清或血浆到一个明确的1.5 毫升管。将其余的材料冷冻在-80 ° c。</li>
	<li>将50µL 的蛋白酶 k (20 毫克/毫升) 直接添加到样品中。</li>
	<li>在样品中加入0.5 毫升的裂解缓冲液, 并由移混合均匀。</li>
	<li>关闭管和孵化样品在56° c 为15分钟的热块。</li>
	<li>在洗涤缓冲器1和2中添加指示的100% 乙醇, 如制造商的说明所示。</li>
	<li>将样品带回室温, 加入0.5 毫升的绝对乙醇, 并由移混合均匀。</li>
	<li>将步骤3.7 中获得的混合物的650µL 加到分离柱上, 并在 6000 x g 处离心1分钟。</li>
	<li>丢弃包含流经的管道, 并将柱放在新的清洁收集管上。重复步骤3.8 和 3.9, 再一次。</li>
	<li>添加500µL 洗涤缓冲器 1, 不润湿柱的边缘和离心在 6000 x g 为1分钟。</li>
	<li>丢弃含有流动的管子, 用新的干净的收集管代替。</li>
	<li>添加500µL 洗涤缓冲器 2, 不润湿柱的边缘和离心机全速 (2万 x g) 为3分钟。</li>
	<li>丢弃含有流动的管子, 用新的干净的收集管代替。</li>
	<li>离心机在全速 (2万 x g) 为3分钟, 以消除任何残留洗涤缓冲区。</li>
	<li>把柱子放到一个干净的1.5 毫升管子里。添加150µL 的洗脱缓冲器, 并等待7分钟, 以确保缓冲区润湿列。</li>
	<li>离心机在 8000 x g 为1分钟。</li>
	<li>将洗从步骤3.16 再次移入同一个柱, 并以全速 (2万 x g) 离心机1分钟, 以确保 DNA 的最大回收率。</li>
</ol>
4. DNA 的量化和稀释
<ol>
	<li>使用分光光度计对 DNA 进行量化。根据制造商的指示, 使用2µL 的样品在台式分光光度计上。 注: 如果 dna 数量不足以进行 real-time PCR (至少 120 ng), 则进行新的 dna 分离过程。</li>
	<li>将血清或血浆中的 DNA 稀释到 2.5 ng/µL, 最终体积足以用于所有 real-time PCR (约50µL)。</li>
</ol>
5. 实时 PCR
<ol>
	<li>解冻拷贝数化验, 参考基因检测, DNA 稀释样品, 和校验仪汇集在冰。平衡在室温下储存在4° c 的主混合。</li>
	<li>准备一个混合1µL 的目标化验, 1 µL 的参考基因检测, 和10µL 的主混合3复制的每个样本和校准仪汇集。准备混合包括负控制 (水) 和2额外的样品。</li>
	<li>在一个96井板, 分12µL 的混合到每一个井。不要吸管或旋转管。</li>
	<li>添加8µL (浓度 2.5 ng/µL) 的每个 DNA 样本和校准仪汇集到每一个井。不要吸管或旋转管。改变每个井的小费。 注:30 DNA 样品可以用96井板进行实时实验处理:30 x 3 样品 + 1 x 3 校验仪池 + 负控制 = 94。</li>
	<li>简要地在 1000 x g 旋转盘子。</li>
	<li>使用以下协议运行板: 举行阶段在95° c 为10分钟, 40 周期在95° c 为十五年代, 和60° c 为 1 min。</li>
</ol>
6. 数据分析和解释
<ol>
	<li>在 "选项" 一节中, 在放大图结果的下面, 将第一个目标基因的 Ct 阈值设置为0.2。为每个目标或参考基因重复该设置。要将 real-time PCR 文件导出为 .txt 扩展名, 请按 "导出" 工具栏。只标记 "结果" 在 "选择要导出的数据" → "选择文件类型" 扩展名. txt →按 "开始导出" →关闭导出工具。</li>
	<li>打开分析软件并导入文件 .txt (工具栏→导入)。</li>
	<li>选择要分析的文件名称, 然后按工具栏 ("播放符号") 选择分析设置。</li>
	<li>指定校准器示例和已知的目标副本数 (例如, 1 副本用于AR基因)。按 "申请"</li>
	<li>对于每个样品, 省略一井与不同的三角洲 Ct (ΔCt) 与其他井比较。</li>
	<li>分析实验 (按播放符号)。</li>
	<li>通过复制数字条形图或表来计算结果。 注意: 结果表包含许多参数, 如置信度、Z 评分和分析的复制, 这对结果解释很有用。</li>
</ol>

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作者声明没有竞争的金融利益。

AR血清和血浆样品中的 CN 分析是一种新的非侵入性方法, 用于去势抗前列腺癌 (不动产) 患者的分层。最近已经证明, AR CN 能够预测不动产患者治疗阿比特龙和 enzalutamide, 化疗前后的结果7,8,9,10。
我们的方法的主要优点是, 该协议是非常简单的执行, 包括只有一个 DNA 隔离过程, 一个 real-time PCR 分析和简单的数据解释。此外, 该测试可以在1工作日内快速完成, 而且程序也很便宜 (大约每样20美元)。我们已经证明, 这种方法是高度重现性的, 并取得的结果是符合那些来自更昂贵和准确的方法, 包括数字 PCR7,8。此外, 我们发现了一个类似的AR增益频率检测与目标 NGS 方法对等离子体样品处理阿比特龙9,10。另一个重要的优势是, 这种方法是灵活的, 可以适用于其他疾病, 其中AR基因具有重要的作用。此外, 还可以根据感兴趣的疾病选择其他目标和参考基因。
CN 分析血清中的AR基因也有一定的局限性。首先, DNA 分光光度定量方法通常不精确, 可以用其他更精确的荧光方法代替。更精确的量化可以提供更精确的 CN 结果。其次, 一些研究表明, 它可以更好地分析血浆 dna 而不是血清 dna。在血清中, 循环细胞游离 dna 的数量一般高于血浆中的12,13, 这是由于血清中白细胞的凝结, 表明血清可能是肿瘤特异 dna 分析的更坏来源因为可能存在的野生型 DNA。
用 real-time PCR 方法对112例不动产患者进行血清中的AR基因的分析, 然后开始治疗 enzalutamide 或阿比特龙。我们的数据显示, 在 cfDNA 上的AR CN 是一个强大的临床结果和抗阿比特龙和 enzalutamide 的基因生物标志物, 并可用于识别可能有益于先验的抗AR疗法或采用快速、低成本的 PCR 方法进行化疗。这些发现突出了研究血液作为一种微创的方法, 在早期和晚期不动产的治疗阻力的机制的效用。未来, 前瞻性和较大的研究应分层患者的循环AR状态, 鉴于随后的临床结果和治疗反应的差异。

血液中的循环细胞游离 DNA (cfDNA) 已被证明是肿瘤诊断、预后、监测和治疗耐药性预测的最理想的生物标志物来源1,2。许多研究表明, 在组织中的 DNA 改变 (突变, 拷贝数变异, 表观修饰) 和对应的血浆样本中发现的1, 证实了循环肿瘤 dna (ctDNA) 的良好一致性初级和转移性肿瘤组织改变的信息3。研究 ctDNA 的可能性因而允许重建基因组重和拷贝数字变异 (cnv) 在具体致癌基因4, 辨认可能的转移的克隆和 subclonal 细胞。CtDNA 已被证明是临床有用的, 特别是癌症治疗监测, 因为它的特定的基因突变和 cnv, 相关的特定靶向治疗5,6。它还克服了组织活检的需要, 并允许在不同的时间在特定的癌症治疗非侵入性的方式获得的结果。
关于前列腺癌, 循环无细胞雄激素受体 (ar) cnv 和治疗反应的阿比特龙和 enzalutamide 的显着相关性已经显示, 表明AR基因复制号 (CN) 在 cfDNA 可能是一个有前途的生物标志物能够预测治疗阻力7,8,9,10,11。利用不同的灵敏度、成本和速度 (e. g、real-time、数字 PCR 和下一代测序), 可以对 ctDNA 中特定基因的 cnv 进行评估。
在这里, 我们描述了一个简单和快速的方法, 基于双相分析的 real-time PCR 技术, 以评估AR CN 在 cfDNA 从血清和血浆样本7,8。我们考虑了两种不同的 PCR 分析设计的两个不同的基因组区域内的内含子5的AR (Xq12) 和其他两个基因, 作为内部标准参考基因已知有一个正常的拷贝数状态在前列腺癌 (RNaseP,位于 14q11;前1, 位于1p34 上。我们选择了两个参考基因, 而不是一个, 以提高精度和灵敏度的结果。每个化验组合 ( AR-assay_1+RNaseP和AR-assay_2+AGO1的组合检测) 的 DNA 数量为 60 ng。三个健康男性的血清或血浆 DNA 样本被汇集并作为校验仪使用。我们考虑了保险丝 #62; 1.5 用于AR增益和 #60; 0.5 用于删除。这种方法的一个主要优点是它是灵活的, 其他基因也可以被评估, 改变标准的内部参考基因, 根据肿瘤类型和特点。

<table><tbody><tr><td>BD Vacutainer Serum Tubes</td><td>Becton Dickinson</td><td>367814</td><td>whole blood tube for serum</td></tr><tr><td>BD Vacutainer EDTA Tubes</td><td>Becton Dickinson</td><td>366643</td><td>whole blood tube for plasma</td></tr><tr><td>Ethanol absolute</td><td>VWR</td><td></td><td>the user could use also other companies</td></tr><tr><td>TaqMan Copy Number assay</td><td>Thermo Fisher Scientific</td><td>4400291</td><td>Pre-designed and validated assays with FAM-dye. We used the followig assay: AR_assay1: hs04107225 adn AR_assay2: hs04511283</td></tr><tr><td>TaqMan Copy Number assay (modified)</td><td>Thermo Fisher Scientific</td><td>4467084</td><td>Pre-designed assay modified with VIC-dye: AGO1: Hs02320401_cn</td></tr><tr><td>TaqMan Copy Number Reference Assay, human, RNase P</td><td>Thermo Fisher Scientific</td><td>4403326</td><td></td></tr><tr><td>TaqMan Universal PCR Master Mix</td><td>Thermo Fisher Scientific</td><td>4326708</td><td>Master mix for Real Time PCR</td></tr><tr><td>QIAamp DNA Mini Kit (50)</td><td>Qiagen</td><td>51304</td><td>DNA extraction kit</td></tr><tr><td>MicroAmp 96-Well Plates</td><td>Thermo Fisher Scientific</td><td>N8010560</td><td>plates for realt time PCR</td></tr><tr><td>NanoDrop 1000 Spectrophotometer</td><td>Thermo Fisher Scientific</td><td>-</td><td>the user could use also other spectrophotometric methods to quantify DNA</td></tr><tr><td>7500 Fast Real-Time PCR System</td><td>Applied Biosystem</td><td>-</td><td>the user could use also other real time instrument</td></tr><tr><td>CopyCaller Software</td><td>Applied Biosystem</td><td>-</td><td>Software for copy number analysis</td></tr></tbody></table>

serum and plasma copy number detection using real-time pcr

血清和血浆细胞游离 DNA (cfDNA) 已被证明是一个信息, 无创的生物标志物的癌症诊断, 预后, 监测和预测的治疗阻力。从雄激素受体 (AR) 基因拷贝数 (CN) 增益是转移性去势抵抗前列腺癌 (mCRPC) 的一个常见事件的假说出发, 我们建议在 cfDNA 中分析这一事件作为一个潜在的预测生物标志物。
我们使用2不同的 real-time PCR 方法和2参考基因 (RNaseP和前1) 在 cfDNA 中评估了AR CN。每种化验组合均使用 60 ng 的 DNA 量。AR利用数字 PCR 技术确定 CN 增益是一种较为准确的方法。CN 变异分析已经被证明在 mCRPC 的设置中对治疗阻力的预测是有帮助的, 但在不同的病人设置中也可以用于其他用途。CN 对 cfDNA 的分析有几个优点: 无创、快速、易于执行, 并从少量的血清或血浆材料开始。

对所有样品进行分光光度法测定的总 cfDNA 浓度, 显示 6.12 ng/µL (范围: 2.00-23.71 ng/µL) 的血清样品的中位数和 3.21 ng/µL (范围: 2.31-8.49 ng/µL) 血浆样品的中位数。通过 real-time PCR 实验, 我们总共分析了115样品。
对AR的高或低增益患者的混合血清 dna 和健康捐献者的 dna 进行了测试灵敏度评估, 这也被用作拷贝数分析的校验仪样本。如图 2所示, ar CN 在0.375% 的患者 dna 中得到充分检测, 其中ar增益与健康的供体 dna 混合, 以获得可检测到的基因增益 (图 2)。因此, 这种方法检测到的拷贝数增益非常低。计算了每位患者的ΔCt 标准偏差 (中等± SD = 0.1, 范围: 0.00-0.36)。我们使用标准偏差值的中点来选择拷贝数增益截止。
我们分析了使用阿比特龙 (n = 53) 治疗的患者的ar的拷贝数, 发现 16 (30.2%) 患者出现ar增益7。同样, 我们分析了 enzalutamide (n = 59) 治疗的患者的ar基因的拷贝数变化, 在 21 (36%) 患者中发现ar增益8。
此外, 我们使用卡普兰-梅尔方法和对数秩测试对AR CN 与临床结局之间的关联进行了评估, 并发现了一个重要的关联。特别是, 阿比特龙治疗病例系列的AR基因增益显示了2.8 与9.5 月的 non-gained 病例的中位进展自由生存 (PFS) (p和 #60; 0.0001)。在具有AR CN 增益 (p和 #60; 0.0001) 的患者中, 还报告了总体生存率较低的 (OS)。
同样, 对于 enzalutamide 病例系列, 我们发现了一个2.4 与4.0 月的患者的中间 PFS, 对于没有增益的患者 (p = 0.0004), 该值为AR增益。此外, 有AR CN 增益的患者的中位数是6.1 与14.1 月, 对于那些没有增益的人 (p = 0.0003)。使用其他方法 (包括数字 pcr (图 3) 确定了从 real-time pcr 方法获得的所有AR CN 增益结果。
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/56502/56502fig1.jpg"> 
图1。工作流和时间线.方法工作流分为不同的步骤和时间。<a href="//ecsource-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/56502/56502fig1large.jpg" target="_blank">请单击此处查看此图的较大版本.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/56502/56502fig2.jpg"> 
图 2.对ar CN 检测方法的灵敏度进行了测试, 使用2不同的池 (样本1和样本 2) 从健康捐献者和从不动产患者获得的 DNA 中提取的ar基因, 混合在不同浓度。患者的 dna 是混合的百分比, 0.375, 0.75, 1.5, 3, 6, 12, 25, 50, 和 100, 关于总 dna。红线: AR CN 增益 (1.5) 的截止。此图已从 chomiak-salvi et al.中修改7
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/56502/56502fig3.jpg"> 
图 3.AR结果比较了 real-time pcr (qPCR) 和数字 pcr (dPCR) 对几种患者 (x 轴) 的疗效。黑线: AR CN 增益 (1.5) 的截止。此图已从 chomiak-salvi et al.中修改7

Watch this Scientific Journal Video about Serum and Plasma Copy Number Detection Using Real-time PCR at JoVE.com

Serum and Plasma Copy Number Detection Using Real-time PCR

这篇手稿描述的拷贝数变异分析进行血清或血浆 DNA 使用 real-time PCR 方法。该方法适用于抗去势前列腺癌患者的耐药性预测, 但也可为其他疾病提供信息。

应用实时 PCR 技术检测血清和血浆拷贝数

Serum and plasma cell free DNA (cfDNA) has been shown as an informative, non-invasive source of biomarkers for cancer diagnosis, prognosis, monitoring, ...

serum-and-plasma-copy-number-detection-using-real-time-pcr

Nanyang Technological University

Research

JoVE Journal

Cancer Research

11.3K 次观看.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. 这篇手稿描述的拷贝数变异分析进行血清或血浆 DNA 使用 real-time PCR 方法。该方法适用于抗去势前列腺癌患者的耐药性预测, 但也可为其他疾病提供信息。

视频: Serum and Plasma Copy Number Detection Using Real-time PCR

Detection of Copy Number Alterations Using Single Cell Sequencing

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and microbial populations. Traditional methods for assessing genetic heterogeneity have been limited by low resolution, low sensitivity, and/or low specificity. Single cell sequencing has emerged as a powerful tool for detecting genetic heterogeneity with high resolution, high sensitivity and, when appropriately analyzed, high specificity. Here we provide a protocol for the isolation, whole genome amplification, sequencing, and analysis of single cells. Our approach allows for the reliable identification of megabase-scale copy number variants in single cells. However, aspects of this protocol can also be applied to investigate other types of genetic alterations in single cells.

Single cell sequencing is an increasingly popular and accessible tool for addressing genomic changes at high resolution. We provide a protocol that uses single cell sequencing to identify copy number alterations in single cells.

单细胞测序拷贝数改变的检测

Detection of genomic changes at single cell resolution is important for characterizing genetic heterogeneity and evolution in normal tissues, cancers, and ...

科研

遗传学

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal gastric mucosa. Recently, we detected CDH1a transcript variant in fresh-frozen tumor tissues obtained from patients with GC. The expression of this variant in tissue samples was investigated by the chip-based digital PCR (dPCR) approach presented here. dPCR offers the potential for an accurate, robust, and highly sensitive measurement of nucleic acids and is increasingly utilized for many applications in different fields. dPCR is capable of detecting rare targets; in addition, dPCR offers the possibility for absolute and precise quantification of nucleic acids without the need for calibrators and standard curves. In fact, the reaction partitioning enriches the target from the background, which improves amplification efficiency and tolerance to inhibitors. Such characteristics make dPCR an optimal tool for the detection of the CDH1a rare transcript.

Detection of a CDH1 Rare Transcript Variant in Fresh-frozen Gastric Cancer Tissues by Chip-based Digital PCR

The manuscript describes a chip-based digital PCR assay to detect a rare CDH1 transcript variant (CDH1a) in fresh-frozen normal and tumor tissues obtained from patients with gastric cancer.

基于芯片的数字 PCR 检测新鲜冷冻胃癌组织中的一个CDH1罕见转录变体

CDH1a, a non-canonical transcript of the CDH1 gene, has been found to be expressed in some gastric cancer (GC) cell lines, whereas it is absent in normal ...

癌症研究

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

数字聚合酶链反应法测定零星家族性腺瘤性息肉病患者的基因变异

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

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 ...

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

Circulating (of cell-free) microRNAs (miRNAs) are released from cells into the blood stream. The amount of specific microRNAs in the circulation has been linked to a disease state and has the potential to be used as disease biomarker. A sensitive and accurate method for circulating microRNA quantification using a dye-based chemistry and droplet digital PCR technology has been recently developed. Specifically, using Locked Nucleic Acid (LNA)-based miRNA-specific primers with a green fluorescent DNA-binding dye in a compatible droplet digital PCR system it is possible to obtain the absolute quantification of specific miRNAs. Here, we describe how performing this technique to assess miRNA amount in biological fluids, such as plasma and serum, is both feasible and effective.

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

微RNA循环使用定量DNA结合染料化学和液滴的数字PCR

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

生物工程

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.

数字 PCR 在急性心肌梗死和心血管疾病中量化循环 MicroRNAs 的临床观察

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

医学

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.

定量实时反转录 PCR 技术对猕猴猴血浆 MicroRNA 水平的绝对量化

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 ...

生物学

人血清中乙型肝炎病毒 DNA 的检测和定量

Real-Time Polymerase Chain Reaction-Based Detection and Quantification of Hepatitis B Virus DNA

Hepatitis B virus (HBV) is a significant cause of liver disease worldwide. It can lead to acute or chronic infections, making individuals highly susceptible to fatal cirrhosis and liver cancer. Accurate detection and quantification of HBV DNA in the blood are essential for diagnosing and monitoring HBV infection. The most common method for detecting HBV DNA is real-time PCR, which can be used to detect the virus and assess the viral load to monitor the response to antiviral therapy. Here, we describe a detailed protocol for the detection and quantification of HBV DNA in human serum or plasma using an IVD-marked real-time PCR-based kit. The kit uses primers and probes that target the highly conserved core region of the HBV genome and can accurately quantify all HBV genotypes (A, B, C, D, E, F, G, H, I, and J). The kit also includes an endogenous internal control to monitor possible PCR inhibition. This assay runs for 40 cycles, and its cutoff is 38 Ct. For the quantification of HBV DNA in clinical samples, a set of 5 quantification standards is provided with the kit. The standards contain known concentrations of HBV-specific DNA that are calibrated against the 4th WHO International Standard for HBV DNA for the nucleic acid test (NIBSC code 10/266). The standards are used to validate the functionality of the HBV-specific DNA amplification and to generate a standard curve, allowing the quantification of HBV DNA in a sample. HBV DNA as low as 2.5 IU/mL was detected using the PCR kit. The high sensitivity and reproducibility of the kit make it a powerful tool in clinical laboratories, aiding healthcare professionals in effectively diagnosing and managing HBV infections.

作者聚焦:乙型肝炎病毒检测的进步和挑战 

Real-time polymerase chain reaction (PCR)-based detection and quantification of hepatitis B virus (HBV) DNA is a sensitive and accurate method for diagnosing and monitoring HBV infection. Here, we present a protocol for HBV DNA detection and the viral load measurement of a sample.