这项研究是由美国国家癌症研究所癌症研究中心院内研究计划美国国立卫生研究院，部分支持。

所描述的协议是采用按照关于使用人体组织样本的美国国立卫生研究院的规则。 1，组织准备<ol><li>前年初，根据机构审查委员会（IRB）的协议得到人体组织标本。 </li><li>选择组织/细胞块的类型和相应的处理方法[福尔马林固定石蜡包埋（FFPE），冷冻，或乙醇固定，石蜡包埋（EFPE）]。福尔马林固定提供了最佳的组​​织学，随后用乙醇固定，并快速冷冻。然而，固定和组织处理方法可影响DNA，RNA和蛋白质的数量和质量的下游分子分析，并应予以考虑。 </li><li>切割组织/细胞块切片上选择的滑动型（玻璃，膜玻璃，或金属框架的膜）。 SIVQ分析同样适用于所有三种类型的幻灯片。请注意，（如下所述）用二甲苯和乙伪盖玻片方法醇不能在金属框架膜幻灯片进行由于滑动，必须在舞台上被颠倒。 </li><li>选择一个化学或免疫组织化学为基础的组织染色，以确定所关注的细胞的背景。需要注意的是染色方法也可影响DNA，RNA和蛋白质的质量和组织的数量。在进行染色与协议前评估生物分子的基准质量测试后的组织中。免疫组织化学（IHC）与民建联15，免疫荧光，快红， 从头红，甲苯胺蓝，苏木精伊红（H＆E）（未发表资料）:SIVQ-LCM已对组织/细胞学载玻片沾满了被执行。 </li></ol> 2。标本成像<ol><li>负载滑动到显微切割工具的机动阶段，并启动相关的软件。选中相应的复选框来指定加载幻灯片的位置，并确保所拍摄的图像文件将被保存为JPEG格式。 </li><li>优化IM通过调整图像的亮度和聚焦在屏幕上，并使用本手动对焦轮或通过解剖仪器的软件年龄质量。 <ol><li>使用解剖仪器的软件中的图像工具箱，适当地将灯泡的亮度和摄像机的增益。示例值有亮度= 60和增益= 220，与扩散器。 </li><li>手动或使用该软件中的自动对焦功能对焦。 </li></ol></li><li>捕捉幻灯片提供一个路线图解剖过程的缩略图概览图像。 <ol><li>对于一个uncoverslipped滑动的最佳的图像质量，利用仪器上的冷凝器下方的扩散器，或者，添加乙醇或二甲苯少量（约30微升）以提高耐火指数（伪盖玻片）。当使用二甲苯，要知道他们必须用有毒的和适当的安全措施，包括使用通风橱和防护实验室外套，护目镜和手套。 </li><li> 请勿在投影片上的LCM帽，直到乙醇或二甲苯溶液中完全清除或盖上的聚合物会被扭曲。 </li></ol></li><li>导航在10X，20X，40X或放大要解剖方面的幻灯片，拍摄的图像。如果有必要，加强与软件，如自动更正（在Microsoft Office图片管理器）图像如前所述22。图像必须被捕获JPEG格式，让他们重新导入到自动选型软件。 </li></ol> 3，算法的图像分析<ol><li>转让从捕获显微切割仪图像到SIVQ文件夹。安装并打开ArcturusXT，自动扫描和SIVQ软件包安装到解剖仪器的电脑上。用于访问SIVQ软件，请联系尤利西斯巴里斯博士（ulysses@med.umich.edu）。 </li><li>开SIVQ并加载所捕获的图像的兴趣（JPEG）。&lt;/ LI&gt; <li>导航到感兴趣的区域和/或调节的显示窗口（视口5＆6）的大小。在SIVQ软件，视口5示出了预处理的图像和视口6示出了后处理的图像16。 </li><li>选择环矢量和环要使用的数字的大小。 </li><li>选择要通过右击视口6抓获其上的谓词图像特征。 </li><li>点击"扫描"来分析图像。 </li><li>采用两个滑动杆调节图像匹配的统计概率。上部杆调整整体矢量特异性，并用于从初始扫描（具有灵敏度由选定的"统计"变量的定义），以排除区域可表示包含过多的区域。相反，较低的滑块被用于增加灵敏度，之后进行的扫描，与被分类为是匹配增加面积的意图。这两个滑块控件的UTI丽泽的"统计"变量作为初始基线灵敏度阈值。 </li><li>保存图像，点击"保存为JPEG"（图像已保存在C :/ vq_test文件夹/图片）。 </li><li>分析与算法的图像。算法分析的输出需要产生一个带注释的图像为它在SIVQ-LCM中使用。目前，SIVQ核心引擎的当前版本是beta版测试，并期望在满负荷生产版本（2014年第一季度上市），将包括一个完整的软件开发工具包（SDK）和应用程序编程接口（API），用于简化集成用户生成的空间滤波器，并与核心环匹配引擎的下游流程的数据处理步骤。该SDK将分发了一套完整的文档。 <ol><li>确保SIVQ热图改变为均匀的红色。 </li></ol></li><li>导出图像。至关重要的是，在p重新嵌入的位置坐标通过使用一个十六进制编辑器从原来的解剖仪器的图像文件头粘贴OST分析JPEG图像。相应的数据可能会"启动图像"标记（0xFF的，为0xD8）和第一"定义量化表"标记（0xFF的，为0xDB）之间发现。 </li></ol> 4。显微切割<ol><li>放置在LCM盖在感兴趣的区域，其中所述图像捕获为SIVQ分析的中心。 </li><li>校准和质量控制（QC）的紫外/红外激光器和优化参数，包括功率，持续时间，激光的位置，和UV切割速度（如制造商推荐的）。之前重新导入所分析的图像进行这些校准。 </li><li>打开AutoScanXT（自动选型软件），并从C :/ vq_test文件夹/图片导入所分析的图像。 </li><li>列车自动选择软件来识别SIVQ注释和创建解剖图。 <ol><li>要创建一个寨宁文件，对"红色漆"的SIVQ分析图像选择感兴趣的四个区域（标记为"蓝色圆圈"）。 </li><li>选择的背景区域（标记为"红方块"），它们不被解剖。 </li><li>单击"分析"按钮，生成培训文件，该文件可以保存供日后使用。 </li></ol></li><li>使用适当的红外（IR）和/或紫外（UV）激光器进行显微解剖。 <ol><li>选定区域复制到"活"的形象。 </li><li>在"microdissect"工具箱中，选择适当的红外捕捉或UV切割按钮。 </li></ol></li><li>剥离完成后，将LCM盖到QC站和捕捉解剖组织/细胞的图像。另一种方法是把帽上之前，将其移动到QC站幻灯片的空白区域，这允许用户在拍摄照片以不同的放大倍率。 </li><li>捕获感兴趣的组织区域的图像显微切割后，进一步评估起重效率。 </li><li>如果所需的细胞已被成功地解剖，点击在ArcturusXT软件的"现阶段"按钮，删除该LCM帽发起的分子提取程序用于下游分析。 </li></ol>

<ol>
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迈克尔·埃默特 - 巴克是美国国立卫生研究院持有的专利，涵盖激光捕获显微切割技术的发明者，并通过美国国家卫生研究院技术转让项目获得特许权为基础的支付。

我们提出了一个协议，用于SIVQ-LCM的应用，以microdissect从FFPE人体乳腺组织免疫染色上皮细胞。使用图像分析算法，如SIVQ，降低了实际操作所需的显微切割过程的时间的量。这对于该领域的潜在的重要进步，因为操作者的时间和精力，通常为所关注的细胞的精确解剖中的限速步骤。在本协议中，我们特别适合我们的程序到ArcturusXT仪器，虽然它很可能将有可能适应SIVQ等算法其他市售的显微仪器，以?...

最初开发于90年代中期，激光捕获显微切割（LCM）使用户通过微观可视化1，2，精确捕捉从组织学组织切片特定的细胞或细胞区域。比较LCM的分子分析与组织擦伤许多研究说明了该方法3-12的值。此外，也有关于可用于观看13,14的技术3视频协议的出版物。然而，尽管它被证明价值，LCM可以是繁琐和费力的，当感兴趣的目标是一个分散的细胞群体中的异质组织切片时，或者当需要进行特定的下游应用，如蛋白质组学大量的细胞。放置在操作人员的负担，导致我们结合一个强大的图像分析算法，以指导对LCM进程15制定LCM半自动夹层的方法。 &lt;P类="j​​ove_content"&gt;在与密歇根大学合作，我们的实验室在NIH扩展先前制定和报告的空间不变的矢量量化（SIVQ）算法的方式，使其到半自动化的组织选择过程中固有的引导显微切割，从而使可用在头脑里的病理学家或生命科学家的工具。空间不变的矢量量化（SIVQ）是一种算法，允许用户简单地"点击"感兴趣的组织学特征来创建一个环载体（谓词图像特征），可以用来搜索整个组织学图像，调整阈值的统计根据需要16-21。由此产生的热图显示匹配的质量初始谓词图像特征，并随后被转换成一个单一的颜色，可以导入到LCM仪器（红色）标注地图。自动选型软件，AutoScanXT，然后用于绘制地图的基础上SIVQ的注解引导从组织样品中的靶细胞的捕获。下面详细的协议描述的实施SIVQ进入显微切割工作流程。

<table border="0" cellpadding="0" cellspacing="0" width="586">
	<tbody>
		<tr height="43">
			<td height="43" style="height:43px;width:216px;">Positive Charged Glass Slides</td>
			<td style="width:84px;">Thermo Scientific</td>
			<td style="width:119px;">4951Plus-001</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Xylenes, ACS reagent, &#8805;98.5% xylenes + ethylbenzene basis&#160;</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">247642</td>
			<td style="width:167px;">CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Ethyl Alcohol, U.S.P. 200 Proof, Anhydrous</td>
			<td style="width:84px;">The Warner-Graham Company</td>
			<td style="width:119px;">6.505E+12</td>
			<td style="width:167px;">CAUTION: PLEASE USE PROPER SAFETY PROCEDURES.</td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">Arcturus CapSure Macro LCM Caps</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">LCM0211</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="64">
			<td height="64" style="height:64px;width:216px;">ArcturusXT Laser Microdissection Instrument</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">ARCTURUSXT</td>
			<td style="width:167px;"></td>
		</tr>
		<tr height="127">
			<td height="127" style="height:127px;width:216px;">AutoScanXT Software</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">An optional image analysis program for the ArcturusXT Laser Microdissection Device. This is software is required for SIVQ-LCM.</td>
		</tr>
		<tr height="169">
			<td height="169" style="height:169px;width:216px;">Spatially Invariant Vector Quantization (SIVQ)</td>
			<td style="width:84px;">University of Michigan</td>
			<td style="width:119px;"></td>
			<td style="width:167px;">This tool suite is publicly available for academic collaborations. For access to the SIVQ algorithm, please contact Dr. Ulysses Balis [Ulysses@med.umich.edu]</td>
		</tr>
	</tbody>
</table>

sivq-lcm protocol for the arcturusxt instrument

SIVQ-LCM是一种新的方法，它可以自动和简化了较为传统的，依赖于用户的激光剥离过程。它的目的是建立一个先进的，快速定制的激光剥离技术平台。在这份报告中，我们描述了图像分析软件空间不变的矢量量化（SIVQ）整合到ArcturusXT仪器。该ArcturusXT系统包含一个红外（IR）和紫外（UV）激光，允许特定的细胞或大面积解剖。主要目标是提高速度，精度，激光剥离，以提高样品通量的重现性。这种新方法有利于动物和在研究和临床工作流程人体组织的显微切割。

一个FFPE人体乳腺组织切片的免疫染色用标准IHC协议23细胞角蛋白AE1/AE3。染色后，将组织切片放置在ArcturusXT阶段，如上述的SIVQ-LCM协议发起。自组织不能盖玻片为显微切割的IHC +染色的细胞可以是难以辨别视觉（ 图1A）。因此，为了提供更好的折射率匹配和改进的图像，二甲苯加入到该组织部分来创建一个伪盖玻片15（ 图1B）。 JPEG图像，然后拍摄的伪盖片?...

Watch this Scientific Journal Video about SIVQ-LCM Protocol for the ArcturusXT Instrument at JoVE.com

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM是一种创新的方法，运用计算机算法，空间不变的矢量量化（SIVQ），驱动激光捕获显微切割（LCM）的过程。该SIVQ-LCM流程大大提高显微切割的速度和精度，以在这两个研究和临床应用中的设置。

为ArcturusXT仪器SIVQ-LCM协议

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

sivq-lcm-protocol-for-the-arcturusxt-instrument

Research

JoVE Journal

Biology

8.6K 次观看.  National Institutes of Health.  SIVQ-LCM是一种创新的方法，运用计算机算法，空间不变的矢量量化（SIVQ），驱动激光捕获显微切割（LCM）的过程。该SIVQ-LCM流程大大提高显微切割的速度和精度，以在这两个研究和临床应用中的设置。 

视频: SIVQ-LCM Protocol for the ArcturusXT Instrument

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

蚊子养育的议定书“（冈比亚按蚊）

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

科研

生物学

Neutrophil Isolation Protocol

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory immune response. Due to their key role in inflammation, neutrophil functions such as locomotion, cytokine production, phagocytosis, and tumor cell combat are extensively studied. To characterize the specific functions of neutrophils, a clean, fast, and reliable method of separating them from other blood cells is desirable for in vitro studies, especially since neutrophils are short-lived and should be used within 2-4 hours of collection. Here, we demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media that is a mixture of sodium metrizoate and Dextran 500. The procedure consists of layering whole blood over the density gradient medium, centrifugation, separation of neutrophil layer, and lysis of residual erythrocytes. Cells are then washed, counted, and resuspended in buffer to desired concentration. When performed correctly, this method has been shown to yield samples of >95% neutrophils with >95% viability.

Neutrophils are among the first cells to arrive on the site of inflammatory immune response, and their functions and mechanisms have been studied extensively in vitro. We demonstrate a standard density gradient separation method to isolate human neutrophils from whole blood using commercially available separation media.

嗜中性粒细胞的分离协议

Neutrophil polymorphonuclear granulocytes (PMN) are the most abundant leukocytes in humans and among the first cells to arrive on the site of inflammatory ...

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as tissue morphogenesis, cell-cell adhesion, or cell death. Drosophila embryos expressing GFP are readily imaged using either stereoscopic or confocal microscopy. A goal of any live-imaging protocol is to minimize detrimental effects such as dehydration and hypoxia. Previous protocols for preparing Drosophila embryos for live-imaging analysis have involved placing dechorionated embryos in halocarbon oil and sandwiching them between a halocarbon gas-permeable membrane and a coverslip1-3. The introduction of compression through mounting embryos in this manner represents an undesirable complication for any biomechanical-based analysis of morphogenesis. Our method, which we call the hanging drop protocol, results in excellent viability of embryos during live imaging and does not require that embryos be compressed. Briefly, the hanging drop protocol involves the placement of embryos in a drop of halocarbon oil that is suspended from a coverslip, which is, in turn, fixed in position over a humid chamber. In addition to providing gas exchange and preventing dehydration, this arrangement takes advantage of the buoyancy of embryos in halocarbon oil to prevent them from drifting out of position during timelapse acquisition. This video describes in detail how to collect and prepare Drosophila embryos for live imaging using the hanging drop protocol. This protocol is suitable for imaging dechorionated embryos using stereomicroscopy or any upright compound fluorescence microscope.

The Preparation of Drosophila Embryos for Live-Imaging Using the Hanging Drop Protocol

A simple, inexpensive, and effective method of preparing Drosophila embryos for live-imaging analysis is presented. Our protocol provides humidity and gas exchange and does not compress the Drosophila embryo. This method is suitable for GFP-based live imaging of Drosophila embryos using a stereomicroscope or upright compound microscope.

编制果蝇实时成像的胚胎使用悬滴协议

Green fluorescent protein (GFP)-based timelapse live-imaging is a powerful technique for studying the genetic regulation of dynamic processes such as ...

A Protocol for the Production of KLRG1 Tetramer

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. KLRG1 exists both as a monomer and as a disulfide-linked homodimer. This well-conserved receptor is found on the most mature and recently activated NK cells as well as on a subset of effector/memory T cells.
Using KLRG1 tetramer as well as other methods, E-, N-, and R-cadherins were identified as KLRG1 ligands. These Ca2+-dependent cell-cell adhesion molecules comprises of an extracellular domain containing five cadherin repeats responsible for cell-cell interactions, a transmembrane domain and a cytoplasmic domain that is linked to the actin cytoskeleton. 
Generation of the KLRG1 tetramer was essential to the identification of the KLRG1 ligands. KLRG1 tetramer is also a unique tool to elucidate the roles cadherin and KLRG1 play in regulating the immune response and tissue integrity.

This protocol describes the production of KLRG1 tetramer, which is a powerful tool for the analysis of KLRG1 ligands.

KLRG1四聚体的生产协议

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

A Protocol for Computer-Based Protein Structure and Function Prediction

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the understanding of their biological role. Although experimental methods can provide detailed information for a small fraction of these proteins, computational modeling is needed for the majority of protein molecules which are experimentally uncharacterized. The I-TASSER server is an on-line workbench for high-resolution modeling of protein structure and function. Given a protein sequence, a typical output from the I-TASSER server includes secondary structure prediction, predicted solvent accessibility of each residue, homologous template proteins detected by threading and structure alignments, up to five full-length tertiary structural models, and structure-based functional annotations for enzyme classification, Gene Ontology terms and protein-ligand binding sites. All the predictions are tagged with a confidence score which tells how accurate the predictions are without knowing the experimental data. To facilitate the special requests of end users, the server provides channels to accept user-specified inter-residue distance and contact maps to interactively change the I-TASSER modeling; it also allows users to specify any proteins as template, or to exclude any template proteins during the structure assembly simulations. The structural information could be collected by the users based on experimental evidences or biological insights with the purpose of improving the quality of I-TASSER predictions. The server was evaluated as the best programs for protein structure and function predictions in the recent community-wide CASP experiments. There are currently &gt;20,000 registered scientists from over 100 countries who are using the on-line I-TASSER server.

Guidelines for computer based structural and functional characterization of protein using the I-TASSER pipeline is described. Starting from query protein sequence, 3D models are generated using multiple threading alignments and iterative structural assembly simulations. Functional inferences are thereafter drawn based on matches to proteins with known structure and functions.

基于计算机的蛋白质结构与功能预测议定书

Genome sequencing projects have ciphered millions of protein sequence, which require knowledge of their structure and function to improve the ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

一个简单的协议提取血细胞从野生毛毛虫

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

Optimized Protocol for Retinal Wholemount Preparation for Imaging and Immunohistochemistry

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ring-supported hydrophilized PTFE membrane to provide structural support to both living and fixed tissue during immunohistochemical processing, which allows for the use of a variety of protocols that would otherwise cause damage to the tissue. First, this is demonstrated with bolus loading of fluorescent markers into living retinal tissue. This method allows for quick visualization of targeted structures, while the membrane support maintains tissue integrity during the injection and allows for easy transfer of the preparation for further imaging or processing.
Second, a procedure for antibody staining in tissue fixed with carbodiimide is described. Though paraformaldehyde fixation is more common, carbodiimide fixation provides a superior substrate for the visualization of synaptic proteins. A limitation of carbodiimide is that the resulting fixed tissue is relatively fragile; however, this is overcome with the use of the supporting membrane. Retinal tissue is used to demonstrate these techniques, but they may be applied to any fragile tissue.

The protocol described here is for structural assessment of a wholemount retinal preparation. This includes descriptions of tissue dissection, mounting onto a hydrophilized polytetrafluoroethylene (PTFE) membrane insert, bolus loading with fluorescent markers, and a comparison of fixation with carbodiimide and paraformaldehyde for immunohistochemical analysis of cellular and synaptic components.

视网膜平片准备成像和免疫组化优化方案

Working with delicate tissue can be a complicating factor when performing immunohistochemical assessment. Here, we present a method that utilizes a ...

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, most notably developmental arrest and reduced metabolism. Global gene expression profiling via RNA-Seq can provide important insights into the transcriptional mechanisms of photoperiodic diapause. The Asian tiger mosquito, Aedes albopictus, is an outstanding organism for studying the transcriptional bases of diapause due to its ease of rearing, easily induced diapause, and the genomic resources available. This manuscript presents a general experimental workflow for identifying diapause-induced transcriptional differences in A. albopictus. Rearing techniques, conditions necessary to induce diapause and non-diapause development, methods to estimate percent diapause in a population, and RNA extraction and integrity assessment for mosquitoes are documented. A workflow to process RNA-Seq data from Illumina sequencers culminates in a list of differentially expressed genes. The representative results demonstrate that this protocol can be used to effectively identify genes differentially regulated at the transcriptional level in A. albopictus due to photoperiodic differences. With modest adjustments, this workflow can be readily adapted to study the transcriptional bases of diapause or other important life history traits in other mosquitoes.

An Experimental and Bioinformatics Protocol for RNA-seq Analyses of Photoperiodic Diapause in the Asian Tiger Mosquito, Aedes albopictus

RNA-Seq analyses are becoming increasingly important for identifying the molecular underpinnings of adaptive traits in non-model organisms. Here, a protocol to identify differentially expressed genes between diapause and non-diapause Aedes albopictus mosquitoes is described, from mosquito rearing, to RNA sequencing and bioinformatics analyses of RNA-Seq data.

实验和生物信息学议定书光周期滞育的亚洲虎蚊的RNA序列分析，<em&gt;白纹伊蚊</em

Photoperiodic diapause is an important adaptation that allows individuals to escape harsh seasonal environments via a series of physiological changes, ...

A Protocol for Using Gene Set Enrichment Analysis to Identify the Appropriate Animal Model for Translational Research

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison techniques resulted in contradictory conclusions regarding the relevance of animal models for translational research. A major reason for the discrepancies between different gene expression analyses is the arbitrary filtering of differentially expressed genes. Furthermore, the comparison of single genes between different species and platforms often is limited by technical variance, leading to misinterpretation of the con/discordance between data from human and animal models. Thus, standardized approaches for systematic data analysis are needed. To overcome subjective gene filtering and ineffective gene-to-gene comparisons, we recently demonstrated that gene set enrichment analysis (GSEA) has the potential to avoid these problems. Therefore, we developed a standardized protocol for the use of GSEA to distinguish between appropriate and inappropriate animal models for translational research. This protocol is not suitable to predict how to design new model systems a-priori, as it requires existing experimental omics data. However, the protocol describes how to interpret existing data in a standardized manner in order to select the most suitable animal model, thus avoiding unnecessary animal experiments and misleading translational studies.

We provide a standardized protocol for the use of gene set enrichment analysis of transcriptomic data to identify an ideal mouse model for translational research. 
This protocol can be used with DNA microarray and RNA sequencing data and can further be extended to other omics data if data are available.

一个协议来使用基因集富集分析以确定合适的动物模型转化型研究

Recent studies that compared transcriptomic datasets of human diseases with datasets from mouse models using traditional gene-to-gene comparison ...

Clock Scan Protocol for Image Analysis: ImageJ Plugins

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a closed or segmented convex-shaped region of interest, leading to the generation of an averaged integral radial pixel-intensity profile. This protocol was originally developed in 2006, as a visual basic 6 script, but as such, it had limited distribution. To address this problem and to join similar recent efforts by others, we converted the original clock scan protocol code into two Java-based plugins compatible with NIH-sponsored and freely available image analysis programs like ImageJ or Fiji ImageJ. Furthermore, these plugins have several new functions, further expanding the range of capabilities of the original protocol, such as analysis of multiple regions of interest and image stacks. The latter feature of the program is especially useful in applications in which it is important to determine changes related to time and location. Thus, the clock scan analysis of stacks of biological images may potentially be applied to spreading of Na+ or Ca++ within a single cell, as well as to the analysis of spreading activity (e.g., Ca++ waves) in populations of synaptically-connected or gap junction-coupled cells. Here, we describe these new clock scan plugins and show some examples of their applications in image analysis.

This paper describes two novel ImageJ plugins for &#39;Clock Scan&#39; image analysis. These plugins expand the functionality of the original visual basic 6 program and, most importantly, make the program available to a large research community by bundling it with the ImageJ free image analysis software package.

用于图像分析的时钟扫描协议:ImageJ插件

The clock scan protocol for image analysis is an efficient tool to quantify the average pixel intensity within, at the border, and outside (background) a ...