笔者想感谢月的标准和控制阳性礼物的国家杯状病毒实验室为疾病控制和预防中心（CDC），并提供试剂在约翰霍普金斯大学公共卫生学院的实验室。

1。粪便样本<ol><li>粪便标本应存放冷冻保存的RNA。使10％的粪便暂停，解冻的粪便样本约0.1克和完成1毫升的PBS。 </li><li>取200μL，以避免反复冻融。储存在-70°C等分</li><li>解冻，并在提取前10分钟离心4000克的等分。 </li></ol> 2。二氧化硅粒子的制备与胍和二氧化硅的提取<ol><li>在室温下，暂停30 g和二氧化硅与DH 2 O完成250毫升。 </li><li>经过24小时的沉淀，去除吸入215毫升上清。新增卫生署2 250毫升，暂停摇动二氧化硅颗粒。 </li><li> 24小时后，取出吸上清，调整的二氧化硅悬浮液的pH值，pH值2.0，使用盐酸（HCl）。二氧化硅悬浮液分装在玻璃BOTtles和釜。 </li></ol> 3。与胍和二氧化硅的提取制备的L6缓冲区<ol><li>在室温下，准备由溶解在0.1米的Tris盐酸，pH值6.4，0.2 M EDTA，pH值8.0的解决方案和11毫升50毫升60克异硫氰酸胍（GuSCN）和1.3Ğ的Triton X-100的L6缓冲区。 </li></ol> 4。与胍和二氧化硅的提取制备的L2缓冲<ol><li>在室温下，准备180克（GuSCN）的异硫氰酸胍溶解在150毫升0.1米三盐酸盐，pH值6.4的L2缓冲。 </li></ol> 5。提取工艺<ol><li>十一月阳性的粪便样品在每个提取，作为阳性对照，DEPC处理水，作为阴性对照，应列入。 </li><li>在微型离心管中混合以下; 1毫升缓冲L6，20μL二氧化硅微粒，添加200μL10％的粪便提取物悬浮。涡短暂离开在室温为15英里N。 </li><li>离心6000克暂停10秒，并用L2缓冲1毫升的沉淀两次，由两个清洗用70％乙醇与丙酮和一个时间。在56°C间加热块干燥5分钟沉淀。 </li><li>水合二氧化硅颗粒的RNA加入50μL无RNase的DH 2 0。 15分钟，加入1μL，RNasin，混合反相管孵育56°C间。 </li><li>离心3全速分钟，在台式离心机，并收集40μL含有RNA的上清液。 </li><li>使用的NanoDrop 2000分光光度计（Thermo Scientific的）量化提取的RNA样品。然后存放于-70℃直至使用，长达6个月。 （ 注：一个更好的方式来保存完整总RNA转换成cDNA）。 </li></ol> 6。替代的提取，使用商业RNA采用QIAamp病毒RNA试剂盒发现在这本小册子省充分说明与凯杰套件ided。简要过程如下： <ol><li>缓冲区的AVL吸取560μL，含1.5 mL管载体的RNA，加入140μL10％的粪便暂停。脉冲涡旋混合15秒。在室温下孵育10分钟。 </li><li>为15秒加560μL乙醇（96-100％）和脉冲涡旋混合样品，然后短暂离心。 </li><li>适用于630μL解决自旋列放置在一个2 ml收集管。离心1分钟6000克;然后放入一个干净的2 mL试管离心柱。 </li><li>洗列，含500μL的Buffer AW1，离心1分钟6000Ğ结合的RNA。在一个干净的2 ml收集管的地方列。 </li><li> 500μL缓冲AW2和离心机全速（20000克或14000转）3分钟，洗净列。 </li><li>放置在一个干净的1.5 ml的微离心管中离心柱，添加40μLDEPC处理过的水洗脱全速的RNA。重复上最终80μL洗脱RNA的CE。 </li><li>使用的NanoDrop 2000分光光度计（Thermo Scientific的）量化提取的RNA样品。然后存放于-70℃直至使用，长达6个月。 （ 注：一个更好的方式来保存完整总RNA转换成cDNA）。 </li></ol> 7。一步与特定的TaqMan探针实时定量RT-PCR技术检测诺沃克病毒中提取的RNA 仪器StepOnePlus实时PCR系统（Applied Biosystems公司）。 方法论<ol><li>擦拭和清洁用RNase所有工作表面，移液器，离心机，以消除任何潜在的RNase污染。 </li><li>吸取11月GI和GII筛选主混合根据表1。引物和TaqMan探针列于表2。 </li><li>适当的高手组合到每个反应管分装10μL。 </li><li>加入5μL稀释的未知样品作为阴性对照的RNA，DEPC处理过的水，或胃肠道分别GII的阳性对照RNA，相应的反应井。所有样品应运行一式两份。阳性对照和标准浓度为300微克/μL和500微克/微升，分别由国家杯状病毒实验室疾病控制和预防中心（CDC）提供。 </li><li>持续10秒离心6000克的反应板。 </li><li>在实验中的属性屏幕;定义和选择运行实验的类型。确保TaqMan试剂的试剂类型，读，预PCR扩增被选中显示。 </li><li>运行15μL的反应，在表3中使用的热剖面。 </li><li>分析结果。 </li></ol> 8。十一月GI和GII使用常规RT-PCR和测序的基因分型 （它没有提到这部影片，因为它是一种常见和广泛使用的方法。） 仪器。应用生物系统公司StepOne扩增和StepOnePlus实时PCR系统与Quantitec模板。 方法论<ol><li>基因分型，前genogroup（GI或GII）的样品RT-PCR方法上面介绍的筛选确定。胃肠月RNA有胃肠基因分型的主结构和全球信息基础设施与全球信息基础设施的基因分型预混月的RNA扩增。 </li><li>吸取11月GI和GII基因分型主混合根据表4。引物胃肠SKF / SKR和G2 SKF / SKR型列于表5。 </li><li>分装45μL适当的主组合，以0.2 ml反应管。 </li><li>加入5μLDEPC处理过的水，每个阴性对照管和5μL预先筛选，NOV（GI和/或GII）的相应的反应管中的积极RNA。 </li><li>运行50μL的反应，在表6中使用的热剖面。 </li><li>发送PCR产物contai宁至少100毫微克/μL，扩增衣壳地区Macrogen公司纯化及测序。 （9700塞内卡大高速公路洛克维尔，MD 20850，每测序样品10元）。 </li></ol> 9。代表结果<img src="/files/ftp_upload/3232/3232fig1.jpg" alt="图1" /> 图1显示的TaqMan一步法RT-PCR技术用于检测RNA的腹泻儿童的粪便样本中提取的具有代表性的结果。循环阈值（CT）为阳性样本被设定在小于或GI和CT 39 GII的平等的CT 37。被发现为阳性对照的Ct值小于27和18，分别为GI和GII的ORF1的ORF2交界。 <a href="http://www.jove.com/files/ftp_upload/3232/3232fig1large.jpg" target="_blank">点击这里查看大图</a> 。 <img src="/files/ftp_upload/3232/3232fig2.jpg" alt="图2" /> 图2 </stroNG&gt;显示Ct值的二氧化硅方法胍和QiAmp的病毒RNA试剂盒的头，头比较。这两项测试的数据属于非常接近平等线（斜率= 1和截距= 0）。回归分析和相关系数证实了这一点。所有阴性对照评估，发现为0，由这两种方法。 <table border="1"><tbody><tr><td> 预混 </td><td> 终浓度 </td><td> 量（μl） </td></tr><tr><td> H 2 0聚合酶链反应</td><td></td><td> 2.875 </td></tr><tr><td> quantitect RT-PCR扩增预混</td><td> 1X </td><td> 6.250 </td></tr><tr><td>逆转录混合</td><td> 1X </td><td> 0.125 </td></tr><tr><td> 50μM的齿轮ŕ底漆</td><td> 1 mM </td><td> 0.250 </td></tr><tr><td> 50微米齿轮F底漆</td><td> 1μM的</td><td> 0.250 </td></tr><tr><td> 10微米环探头</td><td> 1 mM </td><td> 0.125/0.250 * </td></tr><tr><td></td><td></td><td> 10.00 </td></tr></tbody></table> * 0.250μL每个探头用于胃肠道测试，而0.125μL每个探头GII的测试。 表1。Qiagen公司Quantitect主筛选GI和GII的组合（特鲁希略等 ，2006）7。 <table border="1"><tbody><tr><td> 名称 </td><td> 基因型组 </td><td> 使用 </td><td> 序列（5&#39;至3&#39;） </td></tr><tr><td> COG 1R </td><td>胃肠道</td><td>底漆</td><td>铁通AGA公司总部大楼猫猫猫TYAÇ </td></tr><tr><td> COG 1F </td><td>胃肠道</td><td>底漆</td><td> CGY TGG ATG的慢性肾小球肾炎的TTY的CAT GA </td></tr><tr><td> COG 2R </td><td>全球信息基础设施</td><td>底漆</td><td> TCG的ACG的CCA大老山隧道氯乙酸交委会ACA </td></tr><tr><td> COG 2F </td><td>全球信息基础设施</td><td>底漆</td><td>车噶尔BCN ATG的TTY的地带TGG ATG的公司</td></tr><tr><td>环1A </td><td>胃肠道</td><td>探测器</td><td> FAM-AGA TYG海巡署个TCY建GTC CA-BHQ-1 </td></tr><tr><td>环1B </td><td>胃肠道</td><td>探测器</td><td> FAM-AGA TCG的千代田大老山隧道建GTC CA-BHQ-1 </td></tr><tr><td> ring2-TP </td><td>全球信息基础设施</td><td>探测器</td><td>票价调整机制的TGG聚糖GGC GAT的政府总部大楼亚洲空运中心的CT-BHQ-1 </td></tr></tbody></table> 表2。genogroups我，二（荫山等。用于实时定量RT-PCR的引物和探针寡核苷酸，2003）8。 <table border="1"><tbody><tr><td> 步骤 </td><td> 温度（℃） </td><td> 时间（分钟） </td><td></td></tr><tr><td> 1 </td><td> 50 </td><td> 30:00 </td><td> cDNA合成</td></tr><tr><td> 2 </td><td> 95 </td><td> 15:00 </td><td>热启动的Taq聚合酶活化</td></tr><tr><td> 3 </td><td> 95 </td><td> 00:15 </td><td></td></tr><tr><td></td><td> 60 </td><td> 01:00 </td><td> 45X周期</td></tr></tbody></table> 表3。热剖面一步TaqMan实时RT-PCR技术（特鲁希略等 ，2006）7。 <table border="1"><tbody><tr><td> 预混 </td><td> 终浓度 </td><td> 量（μl） </td></tr><tr><td> H 2 0聚合酶链反应</td><td></td><td> 29.50 </td></tr><tr><td> 5X Qiagen公司的RT-PCR缓冲液</td><td> 1X </td><td> 10.00 </td></tr><tr><td> 10毫米dNTP混合液</td><td> 0.4毫米</td><td> 2.00 </td></tr><tr><td>酶混合物</td><td></td><td> 2.00 </td></tr><tr><td> 40U /μL，RNAsin </td><td> 20 U /μL的</td><td> 0.50 </td></tr><tr><td> 10μM的SKF公司</td><td> 0.1微米</td><td> 0.50 </td></tr><tr><td> 10μM的SKR型</td><td> 0.1微米</td><td> 0.50 </td></tr></tbody><tbody><tr><td></td><td></td><td> 45.00 </td></tr></tbody></table> 表4。Qiagen公司的一步法RT-PCR主混合基因型GI和GII。 <table border="1"><tbody><tr><td> 名称 </td><td> 基因型组 </td><td> 使用 </td><td> 序列（5&#39;至3&#39;） </td></tr><tr><td> G1SKF </td><td>胃肠道</td><td>底漆</td><td> 5&#39;-CTG的CCC认证棉酚的TTY多伦多亚洲空运中心的GA - 3 </td></tr><tr><td> G1SKR </td><td>胃肠道</td><td>底漆</td><td> 5&#39;-CCA ACC汽车文建TTR的交谘会的一个 - &#39;3 </td></tr><tr><td> G2SKF </td><td>全球信息基础设施</td><td>底漆</td><td> 5&#39;-CNT GGG AGG的图文影像集团航管贲门癌一个 - 3 </td></tr><tr><td> G2SKR</td><td>全球信息基础设施</td><td>底漆</td><td> 5&#39;-CCR的CCN的GCA的TRH的CCR的TTR的TA的CAT-3 </td></tr></tbody></table> 表5。引物用于扩增地区衣壳地区℃（小岛，2002年） 等。5。 <table border="1"><tbody><tr><td> 步骤 </td><td> 温度（℃） </td><td> 时间（分钟） </td><td></td></tr><tr><td> 1 </td><td> 60 </td><td> 30:00 </td><td> cDNA合成</td></tr><tr><td> 2 </td><td> 96 </td><td> 15:00 </td><td>热启动的Taq聚合酶活化</td></tr><tr><td> 3 </td><td> 94 </td><td> 0时03分0 </td><td></td></tr><tr><td></td><td> 52 </td><td> 01:00 </td><td> 40X周期</td></tr><tr><td></td><td> 72 </td><td> 00:30 </td><td></td></tr><tr><td> 4 </td><td> 72 </td><td> 10:00 </td><td>退火</td></tr></tbody></table> 表6。热的TaqMan一步法RT-PCR技术的个人资料。

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没有利益冲突的声明。

我们使用内部方法从粪便标本中分离核酸的经济，商业QIAmp病毒RNA试剂盒Qiagen公司获得平等的结果，并连同荧光定量RT-PCR技术在我们的实验室开发的，我们可以检测到了11月的广泛属于GI和GII的genogroup的基因型。最近出版的C区协议的基因分型率78％6。往年由于当代十一月株的多样性已在增加，成功率将取决于被测试的样品中地区ç引物的错配。常规诊断法是有用的，因为它消除了放大后的?...

<table border="1"><tbody><tr><td> 试剂名称 </td><td> 公司 </td><td> 目录编号 </td><td> 评论 </td></tr><tr><td>异硫氰酸胍</td><td> Sigma-爱秩序的</td><td> G9277 </td><td></td></tr><tr><td>三盐酸盐</td><td> Sigma-Aldrich公司</td><td> T5941 </td><td></td></tr><tr><td> EDTA的</td><td> Sigma-Aldrich公司</td><td> E5134 </td><td></td></tr><tr><td>二氧化硅</td><td> Sigma-Aldrich公司</td><td> S5631 </td><td></td></tr><tr><td> TRITON X 100 </td><td> BDH问题化学品</td><td> 14530 </td><td></td></tr><tr><td>乙酯</td><td> Sigma-Aldrich公司</td><td> D - 5758 </td><td></td></tr><tr><td>采用QIAamp病毒RNA Mini试剂盒（250） </td><td>凯杰</td><td> 52906 </td><td></td></tr><tr><td>定量TEC探针RT-PCR试剂盒（200） </td><td>凯杰</td><td> 204443 </td><td></td></tr><tr><td> Qiagen公司的一步法RT-PCR试剂盒（200） </td><td>凯杰</td><td> 210212 </td><td></td></tr><tr><td> RNase抑制剂2000台</td><td> A.Biosystems </td><td> N808-0119 </td><td> 2000 unids /瓶</td></tr><tr><td>不粘Rnae免费microfuge管</td><td> Ambion公司</td><td> AM12450 </td><td></td></tr><tr><td>超纯琼脂糖​​1000 </td><td> Invitrogen公司</td><td> 16550-100 </td><td></td></tr></tbody></table>

detection and genogrouping of noroviruses from children's stools by taqman one-step rt-pcr

诺如病毒（NoVs）是在全球所有年龄的人散发性急性胃肠炎暴发的首要原因。他们是在与公共健康的影响类似于轮状病毒的儿童住院的重要原因。 NoVs RNA病毒的伟大的遗传多样性，并有不断涌现的新菌株。五genogroups是与他们的许多基因型和亚型的人类感染的最重要的认可; GI和GII。然而，这两个品种的诊断仍然是个问题，延误诊断和治疗。1，2，3 最常用的方法是从粪便标本中提取的RNA病毒RNA的QIAmp Qiagen公司的商业套件。这种方法结合硅胶膜结合特性，缓冲区控制核糖核酸酶和列提供最佳的RNA结合到一起的microspin速度。这种方法简单，快速和可靠的，是卡尔灭蝇灯，一个由制造商提供的说明中详述的几个步骤。 诺沃克病毒是仅次于轮状病毒腹泻的最常见的原因。诺沃克病毒感染的诊断应该是在腹泻的发病机制研究，以及在疫情或个别腹泻病例。但诺罗病毒的诊断，目前的限制，只是由于缺乏简单的诊断方法的几个中心。这延误诊断和治疗1，2，3。此外，由于国家内部和国家之间的成本和监管运输腐蚀性缓冲区使用这些生产工具包构成的后勤问题。因此，在这个协议中，我们描述的替代，经济，内部方法，这是基于对原有的繁荣等。方法4二氧化硅粒子的核酸结合特性一起使用异硫氰酸胍的抗核酸的性质。 <p class=从粪便标本中，几个RT-PCR技术协议，利用不同的目标NoVs株的检测和genogrouping（GI和GII）“jove_content”&gt;已经制定出来。的共识是，RT-PCR技术使用的TaqMan化学将是最好的分子诊断技术，因为它结合了高吞吐量和易用性，灵敏度高，特异性和重复性。在这里，我们描述了一个检测目标的开放阅读框（ORF1基因）的ORF2交界地区; 11月基因组中最保守的地区，因此最适合用于诊断。为进一步的遗传分析传统的RT-PCR技术，针对的主要蛋白质衣壳（C区）引物最初是由小岛等。5高度可变的N-末端壳详细介绍。从常规PCR测序的PCR产物，使属于GI和GII的genogroups基因型分化。

Watch this Scientific Journal Video about Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR at JoVE.com

Detection and Genogrouping of Noroviruses from Children&#039;s Stools By Taqman One-step RT-PCR

一个一步法RT-PCR方法检测检测和孩子的大便，，利用引物和的TaqMan探针的开放阅读框（ORF1基因）的病毒ORF2交界地区，对诺沃克类病毒基因组中最保守的地区是特定genogroup诺沃克病毒分离鉴定描述。详述一个非商业化，具有成本效益的RNA提取方法。

采用TaqMan从儿童凳诺如病毒的检测和Genogrouping一步法RT-PCR技术

Noroviruses (NoVs) are the leading cause of outbreaks of sporadic acute gastroenteritis worldwide in humans of all ages. They are important cause of ...

detection-genogrouping-noroviruses-from-children-s-stools-taqman-one

Research

JoVE Journal

Medicine

Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

17.1K 次观看.  Universidad Peruana Cayetano Heredia. 一个一步法RT-PCR方法检测检测和孩子的大便，，利用引物和的TaqMan探针的开放阅读框（ORF1基因）的病毒ORF2交界地区，对诺沃克类病毒基因组中最保守的地区是特定genogroup诺沃克病毒分离鉴定描述。详述一个非商业化，具有成本效益的RNA提取方法。

视频: Detection and Genogrouping of Noroviruses from Children's Stools By Taqman One-step RT-PCR

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR (qPCR)

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process including development1, apoptosis2, and cell cycle regulation3. MiRNAs are estimated to regulate the expression of 30% to 90% of human genes4 by binding to their target messenger RNAs (mRNAs)5. Widespread dysregulation of miRNAs has been reported in various diseases and cancer subtypes6. Due to their prevalence and unique structure, these small molecules are likely to be the next generation of biomarkers, therapeutic agents and/or targets.
Methods used to investigate miRNA expression include SYBR green I dye- based as well as Taqman-probe based qPCR. If miRNAs are to be effectively used in the clinical setting, it is imperative that their detection in fresh and/or archived clinical samples be accurate, reproducible, and specific. qPCR has been widely used for validating expression of miRNAs in whole genome analyses such as microarray studies7. The samples used in this protocol were from patients who underwent radical prostatectomy for clinically localized prostate cancer; however other tissues and cell lines can be substituted in. Prostate specimens were snap-frozen in liquid nitrogen after resection. Clinical variables and follow-up information for each patient were collected for subsequent analysis8.
Quantification of miRNA levels in prostate tumor samples. The main steps in qPCR analysis of tumors are: Total RNA extraction, cDNA synthesis, and detection of qPCR products using miRNA-specific primers. Total RNA, which includes mRNA, miRNA, and other small RNAs were extracted from specimens using TRIzol reagent. Qiagen's miScript System was used to synthesize cDNA and perform qPCR (Figure 1). Endogenous miRNAs are not polyadenylated, therefore during the reverse transcription process, a poly(A) polymerase polyadenylates the miRNA. The miRNA is used as a template to synthesize cDNA using oligo-dT and Reverse Transcriptase. A universal tag sequence on the 5' end of oligo-dT primers facilitates the amplification of cDNA in the PCR step. PCR product amplification is detected by the level of fluorescence emitted by SYBR Green, a dye which intercalates into double stranded DNA. Specific miRNA primers, along with a Universal Primer that binds to the universal tag sequence will amplify specific miRNA sequences. 
The miScript Primer Assays are available for over a thousand human-specific miRNAs, and hundreds of murine-specific miRNAs. Relative quantification method was used here to quantify the expression of miRNAs. To correct for variability amongst different samples, expression levels of a target miRNA is normalized to the expression levels of a reference gene. The choice of a gene on which to normalize the expression of targets is critical in relative quantification method of analysis. Examples of reference genes typically used in this capacity are the small RNAs RNU6B, RNU44, and RNU48 as they are considered to be stably expressed across most samples. In this protocol, RNU6B is used as the reference gene.

MicroRNA Detection in Prostate Tumors by Quantitative Real-time PCR qPCR

Quantitative Real Time polymerase chain reaction (qPCR) is a rapid and sensitive method to investigate the expression levels of various microRNA (miRNA) molecules in tumor samples. Using this method expression of hundreds of different miRNA molecules can be amplified, quantified, and analyzed from the same cDNA template.

实时定量PCR（qPCR的microRNA检测在前列腺肿瘤）

MicroRNAs (miRNAs) are single-stranded, 18&#x2013;24 nucleotide long, non-coding RNA molecules. They are involved in virtually every cellular process ...

科研

医学

Cerenkov Luminescence Imaging (CLI) for Cancer Therapy Monitoring

In molecular imaging, positron emission tomography (PET) and optical imaging (OI) are two of the most important and thus most widely used modalities1-3. PET is characterized by its excellent sensitivity and quantification ability while OI is notable for non-radiation, relative low cost, short scanning time, high throughput, and wide availability to basic researchers. However, both modalities have their shortcomings as well. PET suffers from poor spatial resolution and high cost, while OI is mostly limited to preclinical applications because of its limited tissue penetration along with prominent scattering optical signals through the thickness of living tissues.
 Recently a bridge between PET and OI has emerged with the discovery of Cerenkov Luminescence Imaging (CLI)4-6. CLI is a new imaging modality that harnesses Cerenkov Radiation (CR) to image radionuclides with OI instruments. Russian Nobel laureate Alekseyevich Cerenkov and his colleagues originally discovered CR in 1934. It is a form of electromagnetic radiation emitted when a charged particle travels at a superluminal speed in a dielectric medium7,8. The charged particle, whether positron or electron, perturbs the electromagnetic field of the medium by displacing the electrons in its atoms. After passing of the disruption photons are emitted as the displaced electrons return to the ground state. For instance, one 18F decay was estimated to produce an average of 3 photons in water5. 
 Since its emergence, CLI has been investigated for its use in a variety of preclinical applications including in vivo tumor imaging, reporter gene imaging, radiotracer development, multimodality imaging, among others4,5,9,10,11. The most important reason why CLI has enjoyed much success so far is that this new technology takes advantage of the low cost and wide availability of OI to image radionuclides, which used to be imaged only by more expensive and less available nuclear imaging modalities such as PET.
 Here, we present the method of using CLI to monitor cancer drug therapy. Our group has recently investigated this new application and validated its feasibility by a proof-of-concept study12. We demonstrated that CLI and PET exhibited excellent correlations across different tumor xenografts and imaging probes. This is consistent with the overarching principle of CR that CLI essentially visualizes the same radionuclides as PET. We selected Bevacizumab (Avastin; Genentech/Roche) as our therapeutic agent because it is a well-known angiogenesis inhibitor13,14. Maturation of this technology in the near future can be envisioned to have a significant impact on preclinical drug development, screening, as well as therapy monitoring of patients receiving treatments.

Cerenkov Luminescence Imaging CLI for Cancer Therapy Monitoring

Use of Cerenkov Luminescence Imaging (CLI) for monitoring preclinical cancer treatment is described here. This method takes advantage of Cerenkov Radiation (CR) and optical imaging (OI) to visualize radiolabeled probes and thus provides an alternative to PET in preclinical therapeutic monitoring and drug screening.

癌症治疗监测的切伦科夫发光成像（CLI）

In  molecular imaging, positron emission tomography (PET) and optical imaging (OI)  are two of the most important and thus most widely used modalities1-3. ...

Implantation of Inferior Vena Cava Interposition Graft in Mouse Model

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. The long-term clinical results showed excellent patency rates, however, with significant incidence of stenosis. To investigate the cellular and molecular mechanisms of vascular neotissue formation and prevent stenosis development in tissue engineered vascular grafts (TEVGs), we developed a mouse model of the graft with approximately 1 mm internal diameter. First, the TEVGs were assembled from biodegradable tubular scaffolds fabricated from a polyglycolic acid nonwoven felt mesh coated with &#949;-caprolactone and L-lactide copolymer. The scaffolds were then placed in a lyophilizer, vacuumed for 24 hr, and stored in a desiccator until cell seeding. Second, bone marrow was collected from donor mice and mononuclear cells were isolated by density gradient centrifugation. Third, approximately one million cells were seeded on a scaffold and incubated O/N. Finally, the seeded scaffolds were then implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. The implanted grafts demonstrated excellent patency (&#62;90%) without evidence of thromboembolic complications or aneurysmal formation. This murine model will aid us in understanding and quantifying the cellular and molecular mechanisms of neotissue formation in the TEVG.

To improve our knowledge of cellular and molecular neotissue formation, a murine model of the TEVG was recently developed. The grafts were implanted as infrarenal vena cava interposition grafts in C57BL/6 mice. This model achieves similar results to those achieved in our clinical investigation, but over a far shortened time-course.

下腔静脉间置移植物在小鼠模型中植入

Biodegradable scaffolds seeded with bone marrow mononuclear cells (BMCs) are often used for reconstructive surgery to treat congenital cardiac anomalies. ...

Isolation and Immortalization of Patient-derived Cell Lines from Muscle Biopsy for Disease Modeling

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected from skin or muscle biopsy tissue available during the patient&#8217;s diagnostic workup. In this protocol, we describe a technique for live cell isolation from small amounts of muscle or skin tissue for primary cell culture. Additionally, we provide a technique for the immortalization of myogenic cell lines and fibroblast cell lines from primary cells. Once cell lines are immortalized, substantial expansion of patient-derived cells can be achieved. Immortalized cells are amenable to many downstream applications, including drug screening and in vitro correction of the genetic mutation. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

This protocol describes techniques for live cell isolation and primary culture of myogenic and fibroblast cell lines from muscle or skin tissue. A technique for the immortalization of these cell lines is also described. Altogether, these protocols provide a reliable tool to generate and preserve patient-derived cells for downstream applications.

从肌肉活检患者来源的细胞系疾病建模隔离和永生化

The generation of patient-specific cell lines represents an invaluable tool for diagnostic or translational research, and these cells can be collected ...

Establishment of Human Epithelial Enteroids and Colonoids from Whole Tissue and Biopsy

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem cells (ISCs) and progeny that progressively migrate and differentiate toward the tip of the villi. These processes, essential for gastrointestinal homeostasis, have been extensively studied using multiple approaches. Ex vivo technologies, especially primary cell cultures have proven to be promising for understanding intestinal epithelial functions. A long-term primary culture system for mouse intestinal crypts has been established to generate 3-dimensional epithelial organoids. These epithelial structures contain crypt- and villus-like domains reminiscent of normal gut epithelium. Commonly, termed &#8220;enteroids&#8221; when derived from small intestine and &#8220;colonoids&#8221; when derived from colon, they are different from organoids that also contain mesenchyme tissue. Additionally, these enteroids/colonoids continuously produce all cell types found normally within the intestinal epithelium. This in vitro organ-like culture system is rapidly becoming the new gold standard for investigation of intestinal stem cell biology and epithelial cell physiology. This technology has been recently transferred to the study of human gut. The establishment of human derived epithelial enteroids and colonoids from small intestine and colon has been possible through the utilization of specific culture media that allow their growth and maintenance over time. Here, we describe a method to establish a small intestinal and colon crypt-derived system from human whole tissue or biopsies. We emphasize the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

We describe a method to establish human enteroids from small intestinal crypts and colonoids from colon crypts collected from both surgical tissue and biopsies. In this methodological article, we present the culture modalities that are essential for the successful growth and maintenance of human enteroids and colonoids.

从整体组织和活检建立人类上皮Enteroids和Colonoids的

The epithelium of the gastrointestinal tract is constantly renewed as it turns over. This process is triggered by the proliferation of intestinal stem ...

Pancreatic Duct Infusion: An Effective and Selective Method of Drug and Viral Delivery

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, pancreatitis, and pancreatic cancer. All three of these diseases mark active areas of study, not only to develop immediate therapy, but also to better understand their pathophysiology. There are few tools to further these areas of study. Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. The technique requires the intricate dissection of the second portion of the duodenum and ampulla, followed by the occlusion of the bile duct and the cannulation of the pancreatic duct. Although the technique is technically challenging at first, the applications are myriad. Ambiguity in the specifics of the procedure between groups highlighted the need for a standard protocol. This work describes the expression of a green fluorescent protein (GFP) within the pancreas after the pancreatic duct infusion of a viral vector expressing GFP versus a sham surgery. The infusion and therefore expression is specific to the pancreas, without expression present in any other tissue type.

Pancreatic duct infusion is an important technique that can allow for lineage tracing, gene introduction, and cell line-specific targeting. A pancreatic duct infusion technique for drug and viral delivery to pancreatic cells is presented here.

胰腺管输液：药物和病毒输送的有效和选择性方法

The pancreas is a bifunctional organ with both endocrine and exocrine components. A number of pathologies can afflict the pancreas, including diabetes, ...

Isolation of Primary Mouse Hepatocytes for Nascent Protein Synthesis Analysis by Non-radioactive L-azidohomoalanine Labeling Method

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for systemic energy homeostasis. Primary hepatocytes isolated from the murine liver constitute a valuable biological tool to understand the functional properties or alterations occurring in the liver. Herein we describe a method for the isolation and culture of primary mouse hepatocytes by performing a two-step collagenase perfusion technique and discuss their utilization for investigating protein metabolism. The liver of an adult mouse is sequentially perfused with ethylene glycol-bis tetraacetic acid (EGTA) and collagenase, followed by the isolation of hepatocytes with the density gradient buffer. These isolated hepatocytes are viable on culture plates and maintain the majority of endowed characteristics of hepatocytes. These hepatocytes can be used for assessments of protein metabolism including nascent protein synthesis with non-radioactive reagents. We show that the isolated hepatocytes are readily controlled and comprise a higher quality and volume stability of protein synthesis linked to energy metabolism by utilizing the chemo-selective ligation reaction with a Tetramethylrhodamine (TAMRA) protein detection method and western blotting analyses. Therefore, this method is valuable for investigating hepatic nascent protein synthesis linked to energy homeostasis. The following protocol outlines the materials and methods for the isolation of high-quality primary mouse hepatocytes and detection of nascent protein synthesis.

Here, we present a protocol for the isolation of healthy and functional primary mouse hepatocytes. Instructions for detecting hepatic nascent protein synthesis by non-radioactive labeling substrate were provided to help understand the mechanisms underlying protein synthesis in the context of energy-metabolism homeostasis in the liver.

非放射性 l-azidohomoalanine 标记法分离原发性小鼠肝细胞对新生蛋白合成的研究

Hepatocytes are parenchymal cells of the liver and engage multiple metabolic functions, including synthesis and secretion of proteins essential for ...

Handheld Metal Detector Screening for Metallic Foreign Body Ingestion in Children

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a handheld metal detector to detect ingested metallic foreign bodies in children. We propose that by introducing handheld metal detector screening early in the triage process of children with high suspicion of metallic foreign body ingestion, the number of radiographs being ordered to localize the metallic foreign body can be reduced in this radio-sensitive population. The study protocol requires the screening of the participants for history of foreign body ingestion and exclusion of patients with respiratory distress or metallic implants. The patient changes to hospital gown and items that could contain metal like eyeglasses, earrings, pendants, and ornaments are removed. The patient is positioned in the center of the room away from other metallic interferences. The working status of the handheld metal detector is first confirmed by eliciting a positive audio-visual signal. Then the screening is done in an erect position with head in extension to expose the neck, from the level of the chin to the level of the hip joint, to cover the anatomical areas from neck to pelvis in a zig-zag manner both anteriorly and posteriorly. A positive audio-visual signal is carefully noted during the scanning for the presence of metallic foreign body. Relevant radiographs are ordered as per the area detected on the metal detector screening. The handheld metal detector was able to precisely identify all the coins among the ingested metallic foreign bodies in our study. The handheld metal detector could not consistently detect non-coin metallic foreign bodies. This protocol demonstrates the accuracy of handheld metal detector in the identification and localization of coins and coin like metallic foreign bodies.

Here, we present a protocol to use the handheld metal detector, to screen for the presence of ingested metallic foreign bodies in children who present to the pediatric emergency medicine department with a history of foreign body ingestion.

金属异物在儿童体内摄取的手持金属探测器筛选

Coins are the most common ingested metallic foreign bodies among children. The goal of this protocol is to assess the accuracy and feasibility of using a ...

Platelet-based Detection of Nitric Oxide in Blood by Measuring VASP Phosphorylation

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent vasoactive agents, nitric oxide (NO), can also act as a powerful inhibitor of platelet aggregation. Direct NO detection in blood is very challenging due to its high reactivity with cell-free hemoglobin that limits NO half-life to the millisecond range. Currently, NO changes after interventions are only estimated based on measured changes of nitrite and nitrate (members of the nitrate-nitrite-NO metabolic pathway). However precise, these measurements are rather difficult to interpret vis a vis actual NO changes, due to the naturally high baseline nitrite and nitrate levels that are several orders of magnitude higher than expected changes of NO itself. Therefore, the development of direct and simple methods that would allow one to detect NO directly is long overdue. This protocol addresses a potential use of platelets as a highly sensitive NO sensor in blood. It describes initial platelet rich plasma (PRP) and washed platelet preparations and the use of nitrite and deoxygenated red blood cells as NO generators. Phosphorylation of VASP at serine 239 (P-VASPSer239) is used to detect the presence of NO. The fact that VASP protein is highly expressed in platelets and that it is rapidly phosphorylated when NO is present leads to a unique opportunity to use this pathway to directly detect NO presence in blood.

Here, we present a protocol to address the potential use of platelets as a highly sensitive nitric oxide sensor in blood. It describes initial platelet preparation and the use of nitrite and deoxygenated red blood cells as nitric oxide generators.

板基检测血液中一氧化氮的 vasp 磷酸化

Platelets are the blood components responsible for proper blood clotting. Their function is highly regulated by various pathways. One of the most potent ...

Assessment and Characterization of Hyaloid Vessels in Mice

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed regression of hyaloid vessels can be seen in diseases such as persistent hyperplastic primary vitreous (PHPV), leading to an obstructed light path and impaired visual function. Understanding the mechanisms underlying the hyaloid vessel regression may lead to new molecular insights into the vascular regression process and potential new ways to manage diseases with persistent hyaloid vessels. Here we describe the procedures for imaging hyaloid in live mice with optical coherence tomography (OCT) and fundus fluorescein angiography (FFA) and a detailed technical protocol of isolating and flat-mounting hyaloid ex vivo for quantitative analysis. Low-density lipoprotein receptor-related protein 5 (LRP5) knockout mice were used as an experimental model of persistent hyaloid vessels, to illustrate the techniques. Together, these techniques may facilitate a thorough assessment of hyaloid vessels as an experimental model of vascular regression and studies on the mechanism of persistent hyaloid vessels.

This protocol describes both in vivo and ex vivo methods to fully visualize and characterize hyaloid vessels, a model of vascular regression in mouse eyes, using optical coherence tomography and fundus fluorescein angiography for the live imaging and ex vivo isolation and subsequent flat mount of hyaloid for quantitative analysis.

小鼠透明血管的评估与表征

In the eye, the embryonic hyaloid vessels nourish the developing lens and retina and regress when the retinal vessels develop. Persistent or failed ...