我们想感谢L.埃文斯，B.格兰杰，四Rissling，Z SIM，A.古德温，光Hayhurst和Kuch D.在该领域的援助。光Galbreath慷慨地提供从贝拉Coola谷鼠肝脏样本。答:贡萨尔维斯席尔瓦和K.拉森有趣的讨论，这种无创性遗传取样方法的设计作出了贡献。这项工作是由自然科学和工程研究理事会，加拿大发现，和UBC那根个人研究补助金至3月。一位瑞士国家科学基金会博士后研究PBSKP3_128523支持的PH值。进行这项研究是从加拿大不列颠哥伦比亚大学（证书号:A07 - 0126）动物保健协议。

1。发圈套在此之前设立一个圈套，一个理想的位置已确定将在鼠兔栖息地或距骨斜坡。这包括干草桩，这是植被缓存动物在夏末，以及在厕所站点发现的新鲜粪便收集。打包带（长度10 - 50CM）​​条卷起提供了360 °粘表面，并在信封鼠的干草桩（图1）或厕所网站入口处的一个类似Web的方式安排。根据岩石的配置上，一块鱼线可用于支持头发的圈套结构，但这往往是没有必要时，入口非常小（直径30厘米），使用的完整描述鱼线，可以发现在亨利和罗素（2010）3。发网罗尽可能经常检查和头发样品存放在磁带上（图2），然后收集和标记。一旦运回实验室，毛发从粘用无菌镊子的磁带，并转移到低温管，并储存在-20 ° C，直到进一步的处理。集群沿着头发圈套在一起的头发样本被认为是属于一个单一的个体，而集群独立样本被假定为属于不同的个人。在后一种情况下，头发，然后放置在不同的管。这些假设可以在以后测试微基因型数据为基础的DNA指纹图谱和身份的概率计算方法。 2。 DNA的提取由于鼠的头发是非常薄的，并包含一个微小的根灯泡，我们先前已经表明，至少25根头发都需要获得足够量的DNA质量好下游PCR 扩增 3 。我们使用DNA IQ系统（Promega公司，麦迪逊，威斯康星州，美国）和一个稍作修改的版本头发样本进行DNA隔离制造商的指示。首先，在一个微型离心机旋转的头发样本，为了避免脱发，同时开放管。然后孵化的解决方案是准备加入10μl的DTT（1M）和蛋白酶K（18ng/ml）加入10μl混合80μl孵育液的结果在终浓度为0.1米DTT和蛋白酶K孵化1.8ng/ml溶液（100微升）加入样品，并在56 ° C下孵育1小时。在此期间，裂解液准备为每100μL裂解液加入1μL的数码地面电视（1M），导致在最后一个0.01M的DTT浓度。准备裂解液（200μL），然后向样品中加入，7μL的DNA IQ树脂，3秒高速振荡，室温下孵育5分钟。该样本是2秒，然后振荡在磁场的立场，从溶液中分离磁珠时插入。余下的解决方案，然后吸气和丢弃，小心不要去打扰磁性树脂颗粒。样本，然后用100μL的准备裂解液，振荡并返回到磁隔离再次发生的立场。裂解液吸气和丢弃，这一步是使用洗涤缓冲液（100μL）重复三次。样本随后离开干在15分钟的磁场立场。干燥后，100μL洗脱缓冲液加入样品，并在65℃5分钟孵育。该样本是振荡并插入磁立场。该解决方案现在包含洗脱DNA，然后转移到1.5 mL Eppendorf管，在-20 ° C保存，直到进一步的操作。肝脏样本中的DNA提取DNA IQ系统和使用作为阳性对照PCR扩增。 3。 PCR扩增从我们的非侵入性的头发圈套和肝脏样本中获得的DNA，然后用放大了一套常用的分子标记（微卫星，AFLP，线粒体细胞色素b和ZFX / ZFY绝育标记）。 PCR产物均用一个Veriti热循环仪（美国应用生物系统公司，福斯特城，加利福尼亚，美国）在25μL的体积，内含:10 - 20纳克的DNA，引物各0.5微米，10毫米的Tris - HCl（pH值8.3），50毫米1.5毫米氯化钾，氯化镁2，200μMdNTPs，10μ牛血清白蛋白（BSA的; NEB公司，伊普斯维奇，马萨诸塞州，美国）和0.5 U AmpliTaq黄金DNA聚合酶（Applied Biosystems公司）。骑自行车的微卫星位点的参数进行了优化，使用触地循环程序（10分钟95℃，35周期在95 ° C，30秒30秒退火，和45小号72 ° C，在72的最后一步° C下10分钟）。退火温度降低1 ° C，每个周期从60到55℃直至达到第六个周期，在这一点其余的29个周期持续在55 ° C。线粒体片段的循环参数如上所述，但包括35个循环与退火温度50 ° C。扩增片段长度多态性进行了以下的小笠原4所描述的脊椎动物基因组的协议。最后，分子进行性别鉴定是使用HinfI酶切5 ZFX / ZFY位点的PCR - RFLP 。从微，AFLPs和细胞色素b基因序列的PCR产物到ABI 3130xl基因分析仪（美国应用生物系统公司），而消化ZFX / ZFX片段100 bp的阶梯上含有3％的琼脂糖凝胶（New England Biolabs公司）一起运行2.5％的SYBR安全DNA凝胶染色（Invitrogen公司，卡尔斯巴德，美国加利福尼亚州）和可视化，使用的是Red的个人凝胶成像系统（阿尔法INNOTECH公司，美国加利福尼亚州圣莱昂纳多）。微和AFLPs可视化使用GeneMapper v3.7（Applied Biosystems公司）和线粒体DNA色谱使用Sequencher V4.7（基因代码公司，安港，心肌梗死，美国）的可视化。 4。代表性的成果从获得使用我们的非侵入性的圈套的头发样本中提取的DNA用于PCR扩增不同类型的分子标记。作为比较的基础上，DNA提取使用，与我们的头发样本扩增肝脏样本。核微卫星位点，成功地扩增头发和肝脏样本（图3）。虽然信号强度更大的肝脏样本时，这没有一个基因型得分的不利影响（图3）。使用AFLPs（图4），线粒体DNA序列（图5），ZFX / ZFY的分子绝育标记（图6）时，取得了类似的结果。 <img src="/files/ftp_upload/2791/2791fig1.jpg" alt="图1"/> 图1:成立一个非侵入性的头发圈套的例子在干草堆里。卷起的透明包装胶带条被安排在一个类似Web的时尚括干草堆里的入口。这里使用的是一块鱼线，为头发圈套提供更多的支持。 <img src="/files/ftp_upload/2791/2791fig2.jpg" alt="图2"/> 图2:一个成功的头发含有大量的包装胶带粘到头发的圈套。 <img src="/files/ftp_upload/2791/2791fig3.jpg" alt="图3"/> 图3显示一个有代表性的核微色谱（Ocp6）8 Genemapper v3.7（美国应用生物系统公司）。顶部色谱放大DNA从肝组织，是杂合子（358分之354）在该位点。底部色谱代表不同的个体表现出杂合基因型（362分之358）从头发中提取的DNA为基础。虽然从头发样本的色谱信号肝脏样品的比强度较低，基因型得分没有这种信号的差异造成不利影响。 <img src="/files/ftp_upload/2791/2791fig4.jpg" alt="图4"/> 图4。Genemapper v3.7（美国应用生物系统公司）显示一个代表性的AFLP色谱（E31T32）。顶部色谱放大DNA从肝组织中，底部色谱代表从头发中提取的DNA。 <img src="/files/ftp_upload/2791/2791fig5.jpg" alt="图5"/> 图5。一个有代表性的线粒体DNA序列图（细胞色素b）Sequencher V4.7（基因代码公司）显示。顶部色谱放大DNA从肝组织中，底部色谱代表从头发中提取的DNA。 <img src="/files/ftp_upload/2791/2791fig6.jpg" alt="图6"/> 图6。代表的分子绝育标记（ZFX / ZFY），肝脏和头发样本扩增。这种方法依赖于观察，Y染色体拥有在该地区的HinfI酶切位点，位于X染色体上缺乏。因此，雌性产生的432 bp的两个合作迁移的片段，而男性产生432基点，261 bp和171 bp的片段。

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	<li>Kendall, K. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demography+and+Genetic+Structure+of+a+Recovering+Grizzly+Bear+Population.">Demography and Genetic Structure of a Recovering Grizzly Bear Population.</a> J Wildlife Manage. 73, 3-3 (2009).</li><li>Rudnick, J. A., Katzner, T. E., Bragin, E. A., DeWoody, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+non-invasive+genetic+evaluation+of+population+size,+natal+philopatry,+and+roosting+behavior+of+non-breeding+eastern+imperial+eagles+(Aquila+heliaca)+in+central+Asia.">A non-invasive genetic evaluation of population size, natal philopatry, and roosting behavior of non-breeding eastern imperial eagles (Aquila heliaca) in central Asia.</a> Conserv. Genet. 9, 667-667 (2008).</li><li>Henry, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+population+structure+and+ex+situ+representation+of+the+endangered+Amur+tiger.">In situ population structure and ex situ representation of the endangered Amur tiger.</a> Mol Ecol. 18, 3173-3173 (2009).</li><li>Henry, P., Russello, M. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Obtaining+high+quality+DNA+from+elusive+small+mammals+using+low-tech+hair+snares.">Obtaining high quality DNA from elusive small mammals using low-tech hair snares.</a> Eur J Wildlife Res. , (2010).</li><li>Bonin, A., Pompanon, F., Taberlet, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+amplified+fragment+length+polymorphism+(AFLP)+markers+in+surveys+of+vertebrate+diversity.+Method+Enzymol+395.">Use of amplified fragment length polymorphism (AFLP) markers in surveys of vertebrate diversity. Method Enzymol 395.</a> , 145-145 (2005).</li><li>Fontanesi, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sexing+European+rabbits+(Oryctolagus+cuniculus),+European+brown+hares+(Lepus+europaeus)+and+mountain+hares+(Lepus+timidus)+with+ZFX+and+ZFY+loci.">Sexing European rabbits (Oryctolagus cuniculus), European brown hares (Lepus europaeus) and mountain hares (Lepus timidus) with ZFX and ZFY loci.</a> Mol Ecol Resour. 8, 1294-1294 (2008).</li><li>Piggott, M. P., Taylor, A. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Remote+collection+of+animal+DNA+and+its+applications+in+conservation+management+and+understanding+the+population+biology+of+rare+and+cryptic+species.">Remote collection of animal DNA and its applications in conservation management and understanding the population biology of rare and cryptic species.</a> Wildl. Res. 30, 1-1 (2003).</li><li>Banks, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Demographic+monitoring+of+an+entire+species+(the+northern+hairy-nosed+wombat,+Lasiorhinus+krefftii)+by+genetic+analysis+of+non-invasively+collected+material.">Demographic monitoring of an entire species (the northern hairy-nosed wombat, Lasiorhinus krefftii) by genetic analysis of non-invasively collected material.</a> Anim. Conserv. 6, 101-101 (2003).</li><li>Litvaitis, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+range-wide+survey+to+determine+the+current+distribution+of+New+England+cottontails.">A range-wide survey to determine the current distribution of New England cottontails.</a> Wildlife Soc B. 34, 1190-1190 (2006).</li><li>Peacock, M. M., Kirchoff, V. S., Merideth, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+nine+polymorphic+microsatellite+loci+in+the+North+American+pika,+Ochotona+princeps.">Identification and characterization of nine polymorphic microsatellite loci in the North American pika, Ochotona princeps.</a> Mol Ecol Notes. 2, 360-360 (2002).</li></ol>

无创性遗传取样，已成为一个有吸引力的替代传统的生活诱捕方法有以下几个原因。首先，悄悄地收集野生种群的生物材料（如粪便，毛发，羽毛，唾液和粘液），研究人员可以研究这些物种，互不干扰，处理，甚至观察他们，从而降低风险，这两种动物和研究人员。二，非侵入性的遗传采样使生物学家研究难以捉摸和珍稀物种的种群数量，任务可以证明难以与传统现场捕获方法6。第三，NGS可以潜在地增加样本量，减少干扰动物，抽样努力和成本，从而有助于最大限度地减少对人口参数 7的估计偏差。这后一点可能证明是至关重要的处理与受威胁的物种时，由于人口参数的估计偏差可能会导致不适当的管理。 在目前的视频文章中，我们描述了一个简单，新颖，价廉，无创的方法，以样本难以捉摸小型哺乳动物，使用美国皮卡为例。我们从头发中提取的DNA进行同样从肝脏样本中提取DNA微卫星，AFLP，线粒体和绝育标记，使这一非侵入性的抽样方法有吸引力的替代生活诱捕或致命的采样。总体而言，我们预计在罕见或难以实现的小型哺乳动物物种种群的遗传和行为研究的数据收集阶段，这种方法是有用的，同时尽量减少对研究生物的影响。

发圈套: <ul><li>罗尔斯明确的封箱胶带（3M公司，圣保罗，美国明尼苏达州） </li><li>鱼线（10磅） </li><li>低温管（2毫升） </li><li>无菌镊子</li><li>液氮杜瓦（可选） </li></ul> DNA的提取: <ul><li> DNA IQ系统（目录号DC6701; Promega公司）组织和毛发提取试剂盒（目录号DC6740; Promega公司）。 </li><li> MagneSphere科技磁分离架（目录号Z5342; Promega公司） </li><li>微型离心机</li><li>杂交烘箱或水浴</li><li> 1.5 ml Eppendorf管中</li></ul>

a noninvasive hair sampling technique to obtain high quality dna from elusive small mammals

无创性遗传取样方法正变得越来越重要，研究野生动物种群。一些研究报告说，使用非侵入性的抽样技术，调查种群遗传学和人口的1野生种群。这种方法已被证明是与罕见或难以实现的物种2处理时特别有用。虽然这些方法已发展到样品毛发，粪便和其他食肉动物和中型哺乳动物的生物材料，他们大都还停留在难以捉摸的小型哺乳动物未经测试。在这段视频中，我们提出了一个新颖，价格低廉，无创发圈套在一个难以捉摸的小型哺乳动物，美国鼠（兔princeps）针对性。我们描述了一般的头发圈套，包括包装磁带安排在一个类似Web的方式，在鼠兔栖息地的旅游路线沿条设置。我们将举例说明，在收集大量的头发，然后将其收集并带回实验室效率的圈套。然后，我们展示一个使用的DNA IQ系统（Promega公司）提取DNA，并展示了这种方法扩增包括核微卫星，扩增片段长度多态性（AFLPs），线粒体序列（800bp），以及常用的分子标记的实用性分子性别鉴定标记。总的来说，我们表现出的这本小说作为一个野生动物的种群生物学家的采样技术的非侵入性的头发圈套的效用。我们预计，这种方法将适用于各种小型哺乳动物，自然种群内开放地区调查，研究有机体的影响的同时最大限度地减少。

Watch this Scientific Journal Video about A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals at JoVE.com

A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

我们目前的一种无创性的抽样方法，有效地收集从难以捉摸的小型哺乳动物的毛发样本，为美国鼠兔所示。从采样头发中提取的DNA和放大分子标记在野生动物生态学和保护的研究中常用的几种类型，我们证明这种方法的效用。

一种无创性的发采样技术，以获得难以捉摸的小型兽类高质量的DNA

Noninvasive genetic sampling approaches are becoming increasingly important to study wildlife populations. A number of studies have reported using ...

a-noninvasive-hair-sampling-technique-to-obtain-high-quality-dna-from

Research

JoVE Journal

Biology

20.7K 次观看.  University of British Columbia, Okanagan Campus. 我们目前的一种无创性的抽样方法，有效地收集从难以捉摸的小型哺乳动物的毛发样本，为美国鼠兔所示。从采样头发中提取的DNA和放大分子标记在野生动物生态学和保护的研究中常用的几种类型，我们证明这种方法的效用。

视频: A Noninvasive Hair Sampling Technique to Obtain High Quality DNA from Elusive Small Mammals

Obtaining High Quality RNA from Single Cell Populations in Human Postmortem Brain Tissue

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression profiles of pyramidal neurons in layer III. It is well known that the cerebral cortex is an exceptionally heterogeneous structure comprising diverse regions, layers and cell types, each of which is characterized by distinct cellular and molecular compositions and therefore differential gene expression profiles. To circumvent the confounding effects of tissue heterogeneity, we used laser-capture microdissection (LCM) in order to isolate our specific cell-type i.e pyramidal neurons.
Approximately 500 pyramidal neurons stained with the Histogene staining solution were captured using the Arcturus XT LCM system. RNA was then isolated from captured cells and underwent two rounds of T7-based linear amplification using Arcturus/Molecular Devices kits. The Experion LabChip (Bio-Rad) gel and electropherogram indicated good quality a(m)RNA, with a transcript length extending past 600nt required for microarrays. The amount of mRNA obtained averaged 51&mu;g, with acceptable mean sample purity as indicated by the A260/280 ratio, of 2.5. Gene expression was profiled using the Human X3P GeneChip probe array from Affymetrix.

We describe a process using laser-capture microdissection to isolate and extract RNA from a homogeneous cell population, pyramidal neurons, in layer III of the superior temporal gyrus in postmortem human brains. We subsequently linearly amplify (T7-based) mRNA, and hybridize the sample to the Affymetrix human X3P microarray.

人死后脑组织中的单细胞群获得高品质的RNA

We proposed to investigate the gray matter reduction in the superior temporal gyrus seen in schizophrenia patients, by interrogating gene expression ...

科研

生物学

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the richness of biological information it provides. MR does not require any ionizing radiation and can noninvasively provide higher resolution and better signal-to-noise ratio in comparison to other tomographic or spectroscopic modalities. In this protocol, we will focus on small animal MR imaging and MR spectroscopy (MRI/MRS) to noninvasively acquire relaxation weighted 1H images of mouse and to obtain 31P spectra of mouse muscle. This work does not attempt to cover every aspect of small animal MRI/MRS but rather introduces basic procedures of mouse MRI/MRS experiments. The main goal of this work is to inform researchers of the basic procedures for in vivo MR experiments on small animals. The goal is to provide a better understanding of basic experimental procedures to allow researchers new to the MR field to better plan for non-MR components of their studies so that both MR and non-MR procedures are seamlessly integrated.

Noninvasive In Vivo Small Animal MRI and MRS: Basic Experimental Procedures

This work describes basic procedures of noninvasive small animal MRI and MRS in vivo.

无创在体内小动物的MRI和MRS:基本实验程序

Small animal Magnetic Resonance (MR) research has emerged as an important element of modern biomedical research due to its non-invasive nature and the ...

DNA Microarrays: Sample Quality Control, Array Hybridization and Scanning

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, different physiological or pathological states, response to therapy, and, in general, any condition that is being experimentally tested1. Expression profiling of neural tissues requires isolation of high quality RNA, amplification of the isolated RNA and hybridization to DNA microarrays. In this article we describe protocols for reproducible microarray experiments from brain tumor tissue2. We will start by performing a quality control analysis of isolated RNA samples with Agilent's 2100 Bioanalyzer "lab-on-a-chip" technology. High quality RNA samples are critical for the success of any microarray experiment, and the 2100 Bioanalyzer provides a quick, quantitative measurement of the sample quality. RNA samples are then amplified and labeled by performing reverse transcription to obtain cDNA, followed by in vitro transcription in the presence of labeled nucleotides to produce labeled cRNA. By using a dual-color labeling kit, we will label our experimental sample with Cy3 and a reference sample with Cy5. Both samples will then be combined and hybridized to Agilent's 4x44 K arrays. Dual-color arrays offer the advantage of a direct comparison between two RNA samples, thereby increasing the accuracy of the measurements, in particular for small changes in expression levels, because the two RNA samples are hybridized competitively to a single microarray. The arrays will be scanned at the two corresponding wavelengths, and the ratio of Cy3 to Cy5 signal for each feature will be used as a direct measurement of the relative abundance of the corresponding mRNA. This analysis identifies genes that are differentially expressed in response to the experimental conditions being tested.

We demonstrate the use of DNA microarrays for expression profiling of the nervous system. We describe RNA quality control, sample labeling, and array hybridization and scanning.

DNA微阵列:样品的质量控制，阵列杂交和扫描

Microarray expression profiling of the nervous system provides a powerful approach to identifying gene activities in different stages of development, ...

Extraction of High Molecular Weight DNA from Microbial Mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA2,3 during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes4,5,6.
The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 &mu;g of HMW DNA (35-50 kb) per gram of mat sample, with an A260/280 ratio of 1.6. Furthermore, amplification of 16S rRNA genes7 suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.

We provide an improved protocol for extracting high molecular weight DNA from hypersaline microbial mats. Microbial cells are separated from the mat matrix prior to DNA extraction and purification. This enhances the concentrations, quality, and size of the DNA. The protocol may be used for other refractory samples.

超高分子量DNA提取微生物席

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction  of high-quality, high molecular weight ...

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing (ChIP-seq)

ChIP-sequencing (ChIP-seq) methods directly offer whole-genome coverage, where combining chromatin immunoprecipitation (ChIP) and massively parallel sequencing can be utilized to identify the repertoire of mammalian DNA sequences bound by transcription factors in vivo. "Next-generation" genome sequencing technologies provide 1-2 orders of magnitude increase in the amount of sequence that can be cost-effectively generated over older technologies thus allowing for ChIP-seq methods to directly provide whole-genome coverage for effective profiling of mammalian protein-DNA interactions.
For successful ChIP-seq approaches, one must generate high quality ChIP DNA template to obtain the best sequencing outcomes. The description is based around experience with the protein product of the gene most strongly implicated in the pathogenesis of type 2 diabetes, namely the transcription factor transcription factor 7-like 2 (TCF7L2). This factor has also been implicated in various cancers.
Outlined is how to generate high quality ChIP DNA template derived from the colorectal carcinoma cell line, HCT116, in order to build a high-resolution map through sequencing to determine the genes bound by TCF7L2, giving further insight in to its key role in the pathogenesis of complex traits.

Generation of High Quality Chromatin Immunoprecipitation DNA Template for High-throughput Sequencing ChIP-seq

The combination of chromatin immunoprecipitation and ultra-high-throughput sequencing (ChIP-seq) can identify and map protein-DNA interactions in a given tissue or cell line. Outlined is how to generate a high quality ChIP template for subsequent sequencing, using experience with the transcription factor TCF7L2 as an example.

代高通量测序技术（ChIP-seq的高品质的染色质免疫沉淀DNA模板）

ChIP-sequencing (ChIP-seq) methods directly  offer whole-genome coverage, where combining chromatin immunoprecipitation  (ChIP) and massively parallel ...

A Manual Small Molecule Screen Approaching High-throughput Using Zebrafish Embryos

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology due to their considerable degree of genetic conservation with humans. Chemical genetics entails testing the effect that small molecules have on a biological process and is becoming a popular translational research method to identify therapeutic compounds. Zebrafish are specifically appealing to use for chemical genetics because of their ability to produce large clutches of transparent embryos, which are externally fertilized. Furthermore, zebrafish embryos can be easily drug treated by the simple addition of a compound to the embryo media. Using whole-mount in situ hybridization (WISH), mRNA expression can be clearly visualized within zebrafish embryos. Together, using chemical genetics and WISH, the zebrafish becomes a potent whole organism context in which to determine the cellular and physiological effects of small molecules. Innovative advances have been made in technologies that utilize machine-based screening procedures, however for many labs such options are not accessible or remain cost-prohibitive. The protocol described here explains how to execute a manual high-throughput chemical genetic screen that requires basic resources and can be accomplished by a single individual or small team in an efficient period of time. Thus, this protocol provides a feasible strategy that can be implemented by research groups to perform chemical genetics in zebrafish, which can be useful for gaining fundamental insights into developmental processes, disease mechanisms, and to identify novel compounds and signaling pathways that have medically relevant applications.

The zebrafish is an excellent experimental organism to study vertebrate developmental processes and model human disease. Here, we describe a protocol on how to perform a manual high-throughput chemical screen in zebrafish embryos with a whole-mount in situ hybridization (WISH) read-out.

手册小分子屏幕接近高通量使用斑马鱼胚胎

Zebrafish have become a widely used model organism to investigate the mechanisms that underlie developmental biology and to study human disease pathology ...

A New Technique for Quantitative Analysis of Hair Loss in Mice Using Grayscale Analysis

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and alopecia areata. Alopecia can also occur as a side effect of chemotherapy in cancer patients. In this study, our goal was to develop a consistent and reliable method to quantify hair loss in mice, which will allow investigators to accurately assess and compare new therapeutic approaches for these various forms of alopecia. The method utilizes a standard gel imager to obtain and process images of mice, measuring the light absorption, which occurs in rough proportion to the amount of black (or gray) hair on the mouse. Data that has been quantified in this fashion can then be analyzed using standard statistical techniques (i.e., ANOVA, T-test). This methodology was tested in mouse models of chemotherapy-induced alopecia, alopecia areata and alopecia from waxing. In this report, the detailed protocol is presented for performing these measurements, including validation data from C57BL/6 and C3H/HeJ strains of mice. This new technique offers a number of advantages, including relative simplicity of application, reliance on equipment which is readily available in most research laboratories, and applying an objective, quantitative assessment which is more robust than subjective evaluations. Improvements in quantification of hair growth in mice will improve study of alopecia models and facilitate evaluation of promising new therapies in preclinical studies.

Alopecia is a common form of hair loss which can occur in many different conditions, including as a side-effect of chemotherapy. We have developed a method to quantify hair loss in mice, utilizing a standard gel imager to perform a grayscale analysis, to facilitate study of promising new alopecia therapies.

新技术脱发的定量分析小鼠使用灰度分析

Alopecia is a common form of hair loss which can occur in many different conditions, including male-pattern hair loss, polycystic ovarian syndrome, and ...

A Web Tool for Generating High Quality Machine-readable Biological Pathways

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological information. The pathways generated by this online application are machine-readable and fully compatible with essentially all web-browsers and computer operating systems. It uses a specially developed, web-enabled pathway drawing interface that permits the selection and placement of different combinations of pre-drawn biological or biochemical entities to depict reactions, interactions, transport processes and binding events. This palette of entities consists of chemical compounds, proteins, nucleic acids, cellular membranes, subcellular structures, tissues, and organs. All of the visual elements in it can be interactively adjusted and customized. Furthermore, because this tool is a web server, all pathways and pathway elements are publicly accessible. This kind of pathway &#34;crowd sourcing&#34; means that PathWhiz already contains a large and rapidly growing collection of previously drawn pathways and pathway elements. Here we describe a protocol for the quick and easy creation of new pathways and the alteration of existing pathways. To further facilitate pathway editing and creation, the tool contains replication and propagation functions. The replication function allows existing pathways to be used as templates to create or edit new pathways. The propagation function allows one to take an existing pathway and automatically propagate it across different species. Pathways created with this tool can be &#34;re-styled&#34; into different formats (KEGG-like or text-book like), colored with different backgrounds, exported to BioPAX, SBGN-ML, SBML, or PWML data exchange formats, and downloaded as PNG or SVG images. The pathways can easily be incorporated into online databases, integrated into presentations, posters or publications, or used exclusively for online visualization and exploration. This protocol has been successfully applied to generate over 2,000 pathway diagrams, which are now found in many online databases including HMDB, DrugBank, SMPDB, and ECMDB.

PathWhiz is a comprehensive, online pathway drawing tool for generating biochemical and biological pathways. It uses publicly accessible databases and easily expandable palettes consisting of pre-drawn pathway components. This protocol describes how to easily build new pathways, replicate and edit existing pathways, and propagate previously drawn pathways to different organisms.

一个网络工具，用以生成高质量机器可读的生物学途径

PathWhiz is a web server built to facilitate the creation of colorful, interactive, visually pleasing pathway diagrams that are rich in biological ...

A Laser Capture Microdissection Protocol That Yields High Quality RNA from Fresh-frozen Mouse Bones

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser capture microdissection (LCM) procedure. Since LCM studies work with low amounts of material, concerns about limited RNA yields are also important. Therefore, an LCM protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The effect of staining protocol, thickness of cryosections, microdissected tissue quantity, RNA extraction kit, and LCM system used on RNA yield and integrity obtained from microdissected bone cells was evaluated. Eight-&#181;m-thick frozen bone sections were made using an adhesive film and stained using a rapid protocol for a commercial LCM stain. The sample was sandwiched between a polyethylene terephthalate (PET) membrane and the adhesive film. An LCM system that uses gravity for sample collection and a column-based RNA extraction method were used to obtain high quality RNAs of sufficient yield. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study in situ gene expression in cells of any hard tissue in both physiological conditions and disease processes.

A laser capture microdissection (LCM) protocol was developed to obtain sufficient quantity of high-quality RNA for gene expression analysis in bone cells. The current study focusses on mouse femur sections. However, the LCM protocol reported here can be used to study gene expression in cells of any hard tissue.

从新鲜冷冻的小鼠骨骼中产生高质量RNA的激光捕获微解剖协议

RNA yield and integrity are decisive for RNA analysis. However, it is often technically challenging to maintain RNA integrity throughout the entire laser ...

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and calcium handling. For this purpose, atrial or ventricular cardiomyocytes of high quality are necessary to perform patch-clamp measurements or to explore calcium handling abnormalities. However, the limited yield of high-quality cardiomyocytes obtained by current isolation protocols does not allow both measurements in the same mouse. This article describes a method to isolate high-quality murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, for subsequent simultaneous measurements of calcium transients and L-type calcium current from one animal. Mouse hearts are obtained, and the aorta is rapidly cannulated to remove blood. Hearts are then initially perfused with a calcium-free solution (37 &#176;C) to dissociate the tissue at the level of intercalated discs and afterwards with an enzyme solution containing little calcium to disrupt extracellular matrix (37 &#176;C). The digested heart is subsequently dissected into atria and ventricles. Tissue samples are chopped into small pieces and dissolved by carefully pipetting up and down. The enzymatic digestion is stopped, and cells are stepwise reintroduced to physiologic calcium concentrations. After loading with a fluorescent Ca2+-indicator, isolated cardiomyocytes are prepared for simultaneous measurement of calcium currents and transients. Additionally, isolation pitfalls are discussed and patch-clamp protocols and representative traces of L-type calcium currents with simultaneous calcium transient measurements in atrial and ventricular murine myocytes isolated as described above are provided.

Isolation of High Quality Murine Atrial and Ventricular Myocytes for Simultaneous Measurements of Ca2+ Transients and L-Type Calcium Current

Mouse models allow studying key mechanisms of arrhythmogenesis. For this purpose, high quality cardiomyocytes are necessary to perform patch-clamp measurements. Here, a method to isolate murine atrial and ventricular myocytes via retrograde enzyme-based Langendorff perfusion, which allows simultaneous measurements of calcium-transients and L-type calcium current, is described.

分离高质量鼠心房和心室肌细胞，同时测量 Ca2+ 瞬变和 L 型钙电流

Mouse models play a crucial role in arrhythmia research and allow studying key mechanisms of arrhythmogenesis including altered ion channel function and ...