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>

<ol>
	<li>Salvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-free+DNA+as+a+diagnostic+marker+for+cancer:+current+insights.">Cell-free DNA as a diagnostic marker for cancer: current insights.</a> Onco Targets Ther. 9, 6549-6559 (2016).</li><li>Heitzer, E., Ulz, P., Geigl, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+tumor+DNA+as+a+liquid+biopsy+for+cancer.">Circulating tumor DNA as a liquid biopsy for cancer.</a> Clin. Chem. 61 (1), 112-123 (2015).</li><li>Murtaza, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+analysis+of+acquired+resistance+to+cancer+therapy+by+sequencing+of+plasma+DNA.">Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA.</a> Nature. 497 (7447), 108-112 (2013).</li><li>Heitzer, E., Ulz, P., Geigl, J. B., Speicher, M. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+detection+of+genome-wide+somatic+copy+number+alterations+by+liquid+biopsies.">Non-invasive detection of genome-wide somatic copy number alterations by liquid biopsies.</a> Mol. Oncol. 10 (3), 494-502 (2016).</li><li>Diaz, L. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+molecular+evolution+of+acquired+resistance+to+targeted+EGFR+blockade+in+colorectal+cancers.">The molecular evolution of acquired resistance to targeted EGFR blockade in colorectal cancers.</a> Nature. 486 (7404), 537-540 (2012).</li><li>Dawson, S. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+circulating+tumor+DNA+to+monitor+metastatic+breast+cancer.">Analysis of circulating tumor DNA to monitor metastatic breast cancer.</a> N. Engl. J. Med. 368 (13), 1199-1209 (2013).</li><li>Salvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+cell-free+AR+and+CYP17A1+copy+number+variations+may+associate+with+outcome+of+metastatic+castration-resistant+prostate+cancer+patients+treated+with+abiraterone.">Circulating cell-free AR and CYP17A1 copy number variations may associate with outcome of metastatic castration-resistant prostate cancer patients treated with abiraterone.</a> Br. J. Cancer. 112 (10), 1717-1724 (2015).</li><li>Salvi, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circulating+AR+copy+number+and+outcome+to+enzalutamide+in+docetaxel-treated+metastatic+castration-resistant+prostate+cancer.">Circulating AR copy number and outcome to enzalutamide in docetaxel-treated metastatic castration-resistant prostate cancer.</a> Oncotarget. 7 (25), 37839-37845 (2016).</li><li>Romanel, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plasma+AR+and+abiraterone-resistant+prostate+cancer.">Plasma AR and abiraterone-resistant prostate cancer.</a> Sci. Transl. Med. 7 (312), (2015).</li><li>Conteduca, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Androgen+receptor+gene+status+in+plasma+DNA+associates+with+worse+outcome+on+enzalutamide+or+abiraterone+for+castration-resistant+prostate+cancer:+a+multi-institution+correlative+biomarker+study.">Androgen receptor gene status in plasma DNA associates with worse outcome on enzalutamide or abiraterone for castration-resistant prostate cancer: a multi-institution correlative biomarker study.</a> Ann Oncol. , (2017).</li><li>Attard, G., Antonarakis, E. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prostate+cancer:+AR+aberrations+and+resistance+to+abiraterone+or+enzalutamide.">Prostate cancer: AR aberrations and resistance to abiraterone or enzalutamide.</a> Nat. Rev. Urol. 13 (12), 697-698 (2016).</li><li>Lee, T. H., Montalvo, L., Chrebtow, V., Busch, M. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitation+of+genomic+DNA+in+plasma+and+serum+samples:+higher+concentrations+of+genomic+DNA+found+in+serum+than+in+plasma.">Quantitation of genomic DNA in plasma and serum samples: higher concentrations of genomic DNA found in serum than in plasma.</a> Transfusion. 41 (2), 276-282 (2001).</li><li>Chan, K. C., Yeung, S. W., Lui, W. B., Rainer, T. H., Lo, Y. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+preanalytical+factors+on+the+molecular+size+of+cell-free+DNA+in+blood.">Effects of preanalytical factors on the molecular size of cell-free DNA in blood.</a> Clin. Chem. 51 (4), 781-784 (2005).</li></ol>

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?...

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?...

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, ...

The overall goal of this method is to detect the gene copy number variation in serum or plasma samples.This method can help answer key questions in advanced prostate cancer field such as detection of markers useful to predict or monitor the spores reduction.The main advantage of this technique is that it is easy and fast to do.The implications of this technique extend toward therapy of advanced prostate cancer because androgen receptor gene copy number variation is a frequent event.Generally, individuals new to this method will struggle because the concentration and the quality of cell-free DNA is often lower than expected.For serum collection, work from five milliliter samples of whole blood and serum without anticoagulants maintained at four degrees Celsius.Start by centrifuging the samples for 15 minutes at 1, 000 g.The supernatant is the serum.Transfer it into two-milliliter tubes and discard the pellet.Unless the serum is processed immediately, freeze the samples at 80

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Cancer Research

11.3K 次观看.  Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS. The overall goal of this method is to detect the gene copy number variation in serum or plasma samples.This method can help answer key questions in advanced prostate cancer field such as detection of markers useful to predict or monitor the spores reduction.The main advantage of this technique is that it is easy and fast to do.The implications of this technique extend toward therapy of advanced prostate cancer because androgen receptor gene copy number variation is a frequent event.Generally,...

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

Detection of Rare Mutations in CtDNA Using Next Generation Sequencing

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. However, the application of this method is challenging due to its low sensitivity in analyzing the trace amount of ctDNA in the blood. Furthermore, the method may generate false positive and negative results from this sequencing and subsequent analysis. To improve the feasibility and reliability of ctDNA detection in the clinic, here we present a technique which enriches rare mutations for sequencing, Enrich Rare Mutation Sequencing (ER-Seq). ER-Seq can distinguish a single mutation out of 1 x 107 wild-type nucleotides, which makes it a promising tool to detect extremely low frequency genetic alterations and thus will be very useful in studying disease heterogenicity. By virtue of the unique sequencing adapter&#39;s ligation, this method enables an efficient recovery of ctDNA molecules, while at the same time correcting for errors bidirectionally (sense and antisense). Our selection of 1021 kb probes enriches the measurement of target regions that cover over 95% of the tumor-related driver mutations in 12 tumors. This cost-effective and universal method enables a uniquely successful accumulation of genetic data. After efficiently filtering out background error, ER-seq can precisely detect rare mutations. Using a case study, we present a detailed protocol demonstrating probe design, library construction, and target DNA capture methodologies, while also including the data analysis workflow. The process to carry out this method typically takes 1-2 days.

This manuscript describes a technique for detecting mutations of low frequency in ctDNA, ER-Seq. This method is differentiated by its unique use of two-directional error correction, a special background filter, and efficient molecular acquirement.

利用下一代测序检测 CtDNA 中的稀有突变

The analysis of circulating tumor DNA (ctDNA) using next-generation sequencing (NGS) has become a valuable tool for the development of clinical oncology. ...

科研

癌症研究

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 ...

Dried Blood and Serum Spots As A Useful Tool for Sample Storage to Evaluate Cancer Biomarkers

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the starting point to achieve reproducible and reliable results. All samples should be treated in the same way from blood collection to storage. Depending on the analyses to be performed, whole blood and serum samples should be stored at -20 &#176;C or -80 &#176;C until use. Blood/serum samples should also be aliquoted to avoid multiple freeze-thawing. Another important issue is the sample conditions during shipment from one laboratory to another. If dry ice is not available or the shipment takes longer than a few days, alternative approaches are needed. One option is to use filter paper for blood collection. Here, we propose a method for blood and serum sample collection that takes advantage of dried blood spots (DBS) and dried serum spots (DSS). We developed the procedure to extract DNA from DBS for the downstream evaluation of some single nucleotide polymorphisms (SNPs) by real time PCR. We also optimized an ELISA assay starting from proteins eluted from DSS. This method can be used with other ELISA assays or procedures evaluating proteins.

This protocol describes a simple and useful method to store peripheral blood and serum/plasma for downstream analyses such as single nucleotide polymorphism (SNP) evaluation and ELISA assay.

干血和血清斑点作为样品贮存的有用工具, 用于评估肿瘤生物标志物

Blood sample quality is crucial to ensure accurate downstream analyses such as real-time PCR or ELISA. Correct storage of biological materials is the ...

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful immunotherapies. Several studies have indicated that tumor infiltrating myeloid cells (e.g., myeloid-derived suppressor cells (MDSCs) and tumor-associated macrophages (TAMs)) suppress cytotoxicity of CD8+ T cells in the tumor microenvironment, and that targeting these regulatory myeloid cells can improve immunotherapies. Here, we present an in vitro assay system to evaluate immune suppressive effects of monocytic-MDSCs and TAMs on the tumor-killing ability of CD8+ T cells. To this end, we first cultured na&#239;ve splenic CD8+ T cells with anti-CD3/CD28 activating antibodies in the presence or absence of suppressor cells, and then co-cultured the pre-activated T cells with target cancer cells in the presence of a fluorogenic caspase-3 substrate. Fluorescence from the substrate in cancer cells was detected by real-time fluorescence microscopy as an indicator of T-cell induced tumor cell apoptosis. In this assay, we can successfully detect the increase of tumor cell apoptosis by CD8+ T cells and its suppression by pre-culture with TAMs or MDSCs. This functional assay is useful for investigating CD8+ T cell suppression mechanisms by regulatory myeloid cells and identifying druggable targets to overcome it via high throughput screening.

Real Time Detection of In Vitro Tumor Cell Apoptosis Induced by CD8+ T Cells to Study Immune Suppressive Functions of Tumor-infiltrating Myeloid Cells

We describe here a protocol to investigate cytotoxicity of pre-activated CD8+ T cells against cancer cells by detecting apoptotic cancer cells via real-time microscopy. This protocol can investigate mechanisms behind myeloid cell-induced T cell suppression and evaluate compounds aimed at replenishing T cells via blockade of immune suppressive myeloid cells.

cd8+ t 细胞诱导体外肿瘤细胞凋亡的实时检测--研究肿瘤浸润髓内细胞的免疫抑制作用

Potentiation of the tumor-killing ability of CD8+ T cells in tumors, along with their efficient tumor infiltration, is a key element of successful ...

Real-Time Monitoring of Human Glioma Cell Migration on Dorsal Root Ganglion Axon-Oligodendrocyte Co-Cultures

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better understand the molecular mechanisms underlying glioma cell migration, an ability to study the interaction between hGCs and axons within the tumor microenvironment is essential. In order to model this cellular interaction, we developed a mixed culture system consisting of hGCs and dorsal root ganglia (DRG) axon-oligodendrocyte co-cultures. DRG cultures were selected because they can be isolated efficiently and can form the long, extensive projections which are ideal for migration studies of this nature. Purified rat oligodendrocytes were then added on purified rat DRG axons and induced to myelinate. After confirming the formation of compact myelin, hGCs were finally added to the co-culture and their interactions with DRG axons and oligodendrocytes was monitored in real-time using time-lapse microscopy. Under these conditions, hGCs form tumor-like aggregate structures that express GFAP and Ki67, migrate along both myelinated and non-myelinated axonal tracks and interact with these axons through the formation of pseudopodia. Our ex vivo co-culture system can be used to identify novel cellular and molecular mechanisms of hGC migration and could potentially be used for in vitro drug efficacy testing.

Here we present an ex-vivo mixed monolayer culture system for the study of human glioma cell (hGC) migration in real-time. This model provides the ability to observe interactions between hGCs and both myelinated and non-myelinated axons within a compartmentalized chamber.

多端根结核细胞迁移的实时监测

Glioblastoma is one of the most aggressive human cancers due to extensive cellular heterogeneity and the migration properties of hGCs. In order to better ...

Detection of Retrotransposition Activity of Hot LINE-1s by Long-Distance Inverse PCR

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so by a process called retrotransposition wherein they transcribe to form an mRNA intermediate which is then consequently inserted into the genome by reverse transcription. Despite being silent in normal cells, LINE-1s are highly active in different epithelial tumors. De novo LINE-1 insertions can potentially drive tumorigenesis, and hence it is important to systematically study LINE-1 retrotransposition in cancer. Out of ~150 retrotransposition-competent LINE-1s present in the human genome, only a handful of LINE-1 loci, also referred to as &#34;hot&#34; LINE-1s, account for the majority of de novo LINE-1 insertion in different cancer types. We have developed a simple polymerase chain reaction (PCR)-based method to monitor retrotransposition activity of these hot LINE-1s. This method, based on long-distance inverse (LDI)-PCR, takes advantage of 3&#180; transduction, a mechanism by which a LINE-1 mobilizes its flanking non-repetitive region, which can subsequently be used to identify de novo LINE-1 3&#180; transduction events stemming from a particular hot LINE-1.

This article outlines a simple PCR-based assay to monitor the activity of an active LINE-1 retrotransposon and to map de novo retrotranspositions in a given genome. Using the MCF7 cell line, we demonstrate herein how this method can be applied to detect activity of a LINE-1 located at 22q12.1.

长距离反向PCR对热线1的逆变活性的检测

Long interspersed nuclear elements 1 (LINE-1s) are the only family of mobile genetic elements in the human genome that can move autonomously. They do so ...

Combined Use of Tail Vein Metastasis Assays and Real-Time In Vivo Imaging to Quantify Breast Cancer Metastatic Colonization and Burden in the Lungs

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification and testing of novel therapeutic targets that will facilitate the development of better treatments for metastatic disease requires preclinical in vivo models. Demonstrated here is a syngeneic mouse model for assaying breast cancer metastatic colonization and subsequent growth. Metastatic cancer cells are stably transduced with viral vectors encoding firefly luciferase and ZsGreen proteins. Candidate genes are then stably manipulated in luciferase/ZsGreen-expressing cancer cells and then the cells are injected into mice via the lateral tail vein to assay metastatic colonization and growth. An in vivo imaging device is then used to measure the bioluminescence or fluorescence of the tumor cells in the living animals to quantify changes in metastatic burden over time. The expression of the fluorescent protein allows the number and size of metastases in the lungs to be quantified at the end of the experiment without the need for sectioning or histological staining. This approach offers a relatively quick and easy way to test the role of candidate genes in metastatic colonization and growth, and provides a great deal more information than traditional tail vein metastasis assays. Using this approach, we show that simultaneous knockdown of Yes associated protein (YAP) and transcriptional co-activator with a PDZ-binding motif (TAZ) in breast cancer cells leads to reduced metastatic burden in the lungs and that this reduced burden is the result of significantly impaired metastatic colonization and reduced growth of metastases.

The described approach combines experimental tail vein metastasis assays with in vivo live animal imaging to allow real-time monitoring of breast cancer metastasis formation and growth in addition to the quantification of metastasis number and size in the lungs.

结合使用尾静脉转移分析与实时 Vivo 成像，以量化乳腺癌转移殖民化和肺负担

Metastasis is the main cause of cancer-related deaths and there are limited therapeutic options for patients with metastatic disease. The identification ...

Impedance-based Real-time Measurement of Cancer Cell Migration and Invasion

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These acquired abnormalities in complex, multilayered molecular signaling networks induce aberrant cell proliferation and survival, extracellular matrix degradation, and metastasis to distant organs. Approximately 90% of cancer-related deaths are estimated to be caused by the direct or indirect effects of metastatic dissemination. Therefore, it is important to establish a highly reliable, comprehensive system to characterize cancer cell behaviors upon genetic and environmental manipulations. Such a system can give a clear understanding of the molecular regulation of cancer metastasis and the opportunity for successful development of stratified, precise therapeutic strategies. Hence, accurate determination of cancer cell behaviors such as migration and invasion with gain or loss of function of gene(s) allows assessment of the aggressive nature of cancer cells. The real-time measurement system based on cell impedance enables researchers to continually acquire data during a whole experiment and instantly compare and quantify the results under various experimental conditions. Unlike conventional methods, this method does not require fixation, staining, and sample processing to analyze cells that migrate or invade. This method paper emphasizes detailed procedures for real-time determination of migration and invasion of glioblastoma cancer cells.

Cancer is a lethal disease due to its ability to metastasize to different organs. Determining the ability of cancer cells to migrate and invade under various treatment conditions is crucial to assessing therapeutic strategies. This protocol presents a method to assess the real-time metastatic abilities of a glioblastoma cancer cell line.&#160;

基于阻抗的癌细胞迁移和入侵的实时测量

Cancer arises due to uncontrolled proliferation of cells initiated by genetic instability, mutations, and environmental and other stress factors. These ...

Measuring Real-time Drug Response in Organotypic Tumor Tissue Slices

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu constitutes the tumor microenvironment (TME) and can modulate response to therapy in vivo or drug response ex vivo. Conventional cancer drug discovery screens are carried out on cells cultured in a monolayer, a system critically lacking the influence of TME. Thus, experimental systems that integrate sensitive and high-throughput assays with physiological TME will strengthen the preclinical drug discovery process. Here, we introduce ex vivo tumor tissue slice culture as a platform for medium-high-throughput drug screening. Organotypic tissue slice culture constitutes precisely-cut, thin tumor sections that are maintained with the support of a porous membrane in a liquid-air interface. In this protocol, we describe the preparation and maintenance of tissue slices prepared from mouse tumors and tumors from patient-derived xenograft (PDX) models. To assess changes in tissue viability in response to drug treatment, we leveraged a biocompatible luminescence-based viability assay that enables real-time, rapid, and sensitive measurement of viable cells in the tissue. Using this platform, we evaluated dose-dependent responses of tissue slices to the multi-kinase inhibitor, staurosporine, and cytotoxic agent, doxorubicin. Further, we demonstrate the application of tissue slices for ex vivo pharmacology by screening 17 clinical and preclinical drugs on tissue slices prepared from a single PDX tumor. Our physiologically-relevant, highly-sensitive, and robust ex vivo screening platform will greatly strengthen preclinical oncology drug discovery and treatment decision making.&#160;

We introduce a protocol for measuring real-time drug response in organotypic tumor tissue slices. The experimental strategy outlined here provides a platform to carry out medium-high throughput drug screens on tissue slices derived from clinical or mouse tumors in ex vivo conditions.

测量组织肿瘤组织切片的实时药物反应

Tumor tissues are composed of cancerous cells, infiltrated immune cells, endothelial cells, fibroblasts, and extracellular matrix. This complex milieu ...

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 ...