本手稿所描述的这项工作的经费由下列机构提供: 教务长大学院长办公室和医学院和牙科院长办公室, 通过卫生科学中心进行计算创新 (HSCCI);埃里森医学基金会新学者老龄化研究 (AG-NS-0681-10) 资助者在研究设计、数据收集和分析、决定出版或编写手稿方面没有任何作用。

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作者声明他们没有竞争的财政利益。

传统和副本集方法都需要对年代衰老动物进行同步处理。我们包括一种方法, 使动物使用次氯酸钠治疗妊娠成人, 只有受精卵与妊娠成人生存治疗。这些胚胎孵化在液体悬浮和发育停止在第一幼虫阶段 (L1)。在将 L1 动物播种到食物上后 (如大肠杆菌表达 dsRNA 的基因), 动物恢复发育。用次氯酸盐治疗妊娠 L1 动物, 可以使残留的非种子选手 L1 动物在液氮或-80 摄氏度冷藏库中无限期地冷冻?...

在了解衰老的遗传基础方面, 最具变革性的技术进步之一是在1线虫中发展基于喂养的 rna 干扰;在实验性地使用 rna 干扰之前, 许多衰老的表型不是遗传的。以喂养为基础的 rna 干扰是通过生产大肠杆菌中的 dsRNA 与内源c. 线虫mRNA: IPTG 诱导双向转录跨插入的任何c. 线虫cDNA 或一部分的在质粒2中打开阅读框架。当C. 线虫饲料在完整的大肠杆菌,由细菌产生的 dsRNA 通过 SID-2 跨膜蛋白3从流明传输到肠道细胞, 然后通过 SID-14分布到其余的动物。在每个细胞内, 外源 dsRNA 由 Dicer 复合体处理成 siRNA, 通过互补基配对与成熟的 mRNA 相互作用, 创建一个新的 siRNA-mRNA 双工。这种双工是由 RISC 复合体和劈裂的, 从而降解内源 mRNA5。因此, 仅仅改变质粒的插入, 你就可以使几乎任何基因在线虫基因组中的功能失效。这一发现导致了几个大的基于喂食的 rna 干扰库的创建--转化的大肠杆菌种群的集合, 可以结合起来以达到约86% 已知的C. 线虫基因的覆盖率6,7。
自从饲养基 rna 干扰的推进, 在C. 线虫的全面屏幕导致发现了900多个基因, 改变生命的灭活时 (由 WormBase 的 rna 干扰-表型协会证明), 我们提到为 gerogenes。大多数 gerogenes 在长寿控制中的作用是在一些精算报告中发现的, 通过喂食为基础的 rna 干扰 (见图 1A和补充文件 1的细节)。在某些情况下, 这些 gerogenes 是根据在单个或几个时间点测量生存能力而确定的, 这不能为使用 rna 干扰治疗的寿命变化提供可量化的衡量标准。在其他情况下, 这些基因已经定量地评估了寿命的变化, 以及其他年龄相关的表型。例如, 我们以前确定了159种基因, 对正常和增加的动物寿命都是必要的, insulin/IGF-1 信号减少, healthspan 的量化变化。其中, 103 基因 inactivations 导致 progeric 表型, 因为损失导致一个或多个过早衰老的迹象8。
虽然有些 gerogenes 与100项或更多的研究相关 (例如 daf-16、daf-2、sir-2.1), 但超过 400 gerogenes 有10或更少的引文 (图 1B和补充文件 2)。因此, 虽然综合饲料基 rna 干扰屏幕发现和马虎的特点数以百计的假定 gerogenes, 如何这些基因在长寿控制中的作用, 这些基因产品之间的遗传关系仍然很差研究。对年龄相关表型的全纵向分析是确定 gerogenes 之间的基因相互作用 (如上位相互作用、asynthetic 相互作用等) 的先决条件。深入了解 gerogenes 之间的遗传相互关系需要一种高通量定量方法, 这也利用了基于喂养的 rna 干扰的优势。
衰老最常见的替代措施是寿命。传统的测量C. 线虫死亡率的方法是在一小部分人口样本中追踪个别动物的死亡情况。随着时间的推移, 动物数量相对较少, 并且周期性地用铂丝或睫毛轻轻地戳, 以运动作为生存能力的指示器 (图 2A)。这种方法得到了广泛的应用, 因为它提供了直接的平均值和最大寿命的测量。然而, 这种传统的方法是耗时和相对较低的吞吐量, 这限制了动物的数量和条件, 可以同时测量的控制方式。最近的一项模拟研究发现, 许多C. 线虫寿命研究并没有对足够数量的动物进行检测, 从而能够可靠地检测出条件9之间的细微变化。此外, 这种传统的方法包括反复处理同一队列动物随着时间的推移, 这反过来可以引入污染, 并可能损害或杀死越来越脆弱, 衰老的动物。
我们开发了一种替代的 "复制集" 方法来测量C. 线虫的寿命。为此, 大量人口的年龄同步, 等基因系动物被划分成少数人口 (或复制品)。生成的副本样本足以覆盖计划实验中的每个时间点。在每个观察时间点, 其中一个副本是为生存, 死亡和被截断的动物的数量得分, 然后动物在复制被丢弃。因此, 在整个人口的预期寿命内, 一系列独立的亚群定期取样 (图 2B)。在使用副本集, 没有重复的刺激动物, 并没有重复暴露在潜在的环境污染。在同一时间点观察到的生存能力完全独立于其他所有观察, 这将最大限度地减少处理和提高吞吐量。这使得我们能够定量8,10的上百个 rna 克隆的寿命变化。
在这里, 我们提出了通过复制集和传统的方法来进行c. 线虫寿命的详细协议, 以评分c. 线虫长寿。我们证明了相似的结果在方法之间得到。我们开发了软件, 以协助对通过任一方法生成的寿命数据进行图形分析, 我们可以根据 GPL V3 许可证免费提供这些信息 (见材料表)。"WormLife" 是在 R11中编写的, 包括图形用户界面 (GUI), 用于绘制数据, 该接口已在 Mac OS 和 Linux 中进行了测试。最后, 我们比较和对比了每种方法的局限性, 并强调在选择方法来测量线虫寿命的定量变化的其他因素。

<table border="0" cellpadding="0" cellspacing="0" width="1120">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">IPTG (isopropyl beta-D-1-thigalactopyranoside)</td>
			<td style="width:108px;">Gold Bio</td>
			<td style="width:119px;">12481C100</td>
			<td style="width:527px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">FuDR (5-Fluoro-2&#39;-deoxyuridine)</td>
			<td style="width:108px;">Alfa Aesar</td>
			<td>L16497</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:367px;">24 Well Culture Plates</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td style="width:119px;">#662102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Retangular non-treated single-well plate, 128x86mm</td>
			<td style="width:108px;">Thermo-Fisher</td>
			<td style="width:119px;">242811</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">600 &#181;L 96-well plates</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td style="width:119px;">#786261</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:367px;">2mL 96-well plates</td>
			<td style="width:108px;">Greiner Bio-One</td>
			<td style="width:119px;">#780286</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Air-permeable plate seal</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">60941-086</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">96-pin plate replicator</td>
			<td style="width:108px;">Nunc</td>
			<td style="width:119px;">250520</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">bacto-peptone</td>
			<td style="width:108px;">VWR</td>
			<td style="width:119px;">90000-368</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">bacteriological agar</td>
			<td style="width:108px;">Affymetrix/USB</td>
			<td style="width:119px;">10906</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:367px;">C. elegans RNAi clone library in HT115 bacteria- Ahringer</td>
			<td style="width:108px;">Source Bioscience</td>
			<td style="width:119px;">C. elegans RNAi Collection (Ahringer)</td>
			<td>See also Kamath et. al, Nature 2003.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:367px;">C. elegans RNAi clone library in HT115 bacteria- Vidal</td>
			<td style="width:108px;">Source Bioscience</td>
			<td style="width:119px;">C. elegans ORF-RNAi Resource (Vidal)</td>
			<td>See also Rual et. al, Genome Research 2004. This library is also available from Dharmacon.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">WormLife- Software for Replica Set Survival Analysis</td>
			<td style="width:108px;">Samuelson Lab</td>
			<td style="width:119px;">N/A</td>
			<td>https://github.com/samuelsonlab-urmc/wormlife</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">L4440 Empty Vector Plasmid</td>
			<td style="width:108px;">Addgene</td>
			<td style="width:119px;">1654</td>
			<td>https://www.addgene.org/1654/</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Wormbase</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>http://www.wormbase.org/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">OASIS</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>https://sbi.postech.ac.kr/oasis2/&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:367px;">Graphpad Prism</td>
			<td style="width:108px;"></td>
			<td style="width:119px;"></td>
			<td>https://www.graphpad.com/scientific-software/prism/&#160;</td>
		</tr>
	</tbody>
</table>

the replica set method: a high-throughput approach to quantitatively measure caenorhabditis elegans lifespan

复制集方法是一种以高通量方式定量测量秀丽线虫的寿命或存活的方法, 从而使单个调查人员能够在相同数量的时间内筛查更多的治疗或条件。没有数据质量损失的时间。这种方法需要在大多数实验室中发现的常见的设备与C. 线虫, 因此是简单的采用。该方法的中心是在每个观察点上对一个种群的独立样本进行化验, 而不是随着时间的推移与传统的纵向方法进行抽样。评分需要在多井板块的井中加入液体, 这会刺激C 线虫移动并促进量化 healthspan 的变化。复制集方法的其他主要好处包括减少琼脂表面暴露于空气中的污染物 (例如霉菌或真菌), 对动物的最小处理, 以及对零星错误评分的鲁棒性 (例如, 当动物仍然活着)。为了对副本集样式实验中的数据进行适当的分析和可视化, 还开发了一种自定义软件工具。该软件的当前功能包括绘制副本集和传统 (卡普兰) 实验的生存曲线, 以及副本集的统计分析。这里提供的协议描述了传统的实验方法和副本集方法, 以及对相应数据分析的概述。

在任何新的方法的发展中, 新方法概括从以前的方法中接受的结果, 并符合领域内的标准势在必行。我们以前的经验表明, 复制集和传统的测定C. 线虫寿命的方法产生类似的结果20。野生型线虫(N2) 维持在20摄氏度通常生活在20和25天之间, 我们观察到的传统 (图 4A, 黑线) 和副本集方法 (图 4B, 黑色)。?...

Watch this Scientific Journal Video about The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan at JoVE.com

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

在这里, 我们描述了复制集方法, 一种定量测量C. 线虫寿命/生存和 healthspan 的方法, 以高通量和健壮的方式, 从而允许筛选许多条件而不牺牲数据质量。该协议详细介绍了策略, 并提供了用于分析副本集数据的软件工具。

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

The Replica Set method is an approach to quantitatively measure lifespan or survival of Caenorhabditis elegans nematodes in a high-throughput manner, thus ...

the-replica-set-method-high-throughput-approach-to-quantitatively

Universidad de Cartagena UDEC

Research

JoVE Journal

Genetics

The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

8.5K 次观看.  University of Rochester Medical Center. 在这里, 我们描述了复制集方法, 一种定量测量C. 线虫寿命/生存和 healthspan 的方法, 以高通量和健壮的方式, 从而允许筛选许多条件而不牺牲数据质量。该协议详细介绍了策略, 并提供了用于分析副本集数据的软件工具。

视频: The Replica Set Method: A High-throughput Approach to Quantitatively Measure Caenorhabditis elegans Lifespan

A Robotic Platform for High-throughput Protoplast Isolation and Transformation

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal transduction pathways, transcriptional regulatory networks, gene expression, genome-editing, and gene-silencing. Furthermore, significant progress has been made in the regeneration of plants from protoplasts, which has generated even more interest in the use of these systems for plant genomics. In this work, a protocol has been developed for automation of protoplast isolation and transformation from a &#39;Bright Yellow&#39; 2 (BY-2) tobacco suspension culture using a robotic platform. The transformation procedures were validated using an orange fluorescent protein (OFP) reporter gene (pporRFP) under the control of the Cauliflower mosaic virus 35S promoter (35S). OFP expression in protoplasts was confirmed by epifluorescence microscopy. Analyses also included protoplast production efficiency methods using propidium iodide. Finally, low-cost food-grade enzymes were used for the protoplast isolation procedure, circumventing the need for lab-grade enzymes that are cost-prohibitive in high-throughput automated protoplast isolation and analysis. Based on the protocol developed in this work, the complete procedure from protoplast isolation to transformation can be conducted in under 4 hr, without any input from the operator. While the protocol developed in this work was validated with the BY-2 cell culture, the procedures and methods should be translatable to any plant suspension culture/protoplast system, which should enable acceleration of crop genomics research.

A high-throughput, automated, tobacco protoplast production and transformation methodology is described. The robotic system enables massively parallel gene expression and discovery in the model BY-2 system that should be translatable to non-model crops.

机器人平台的高通量原生质体分离和转型

Over the last decade there has been a resurgence in the use of plant protoplasts that range from model species to crop species, for analysis of signal ...

科研

遗传学

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

核密度分析的天然染色质沉淀测序文库的生成

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the intestine the primary location for disease. The nematode intestine structurally and functionally mimics mammalian intestines and is transparent making it amenable to microscopic study of colonization. Here we show that pathogens can cause disease and death. We are able to identify microbial mutants that show altered virulence. Its conserved innate response to biotic stresses makes C. elegans an excellent system to probe facets of host innate immune interactions. We show that hosts with mutations in the dual oxidase gene cannot produce reactive oxygen species and are unable to resist microbial insult. We further demonstrate the versatility of the presented survival assay by showing that it can be used to study the effects of inhibitors of microbial growth. This assay may also be used to discover fungal virulence factors as targets for the development of novel antifungal agents, as well as provide an opportunity to further uncover host-microbe interactions. The design of this assay lends itself well to high throughput whole-genome screens, while the ability to cryo-preserve worms for future use makes it a cost-effective and attractive whole animal model to study.

The Nematode Caenorhabditis Elegans - A Versatile In Vivo Model to Study Host-microbe Interactions

Here, we present the nematode Caenorhabditis elegans as a versatile host model to study microbial interaction.

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

We demonstrate a method using Caenorhabditis elegans as a model host to study microbial interaction. Microbes are introduced via the diet making the ...

Robust DNA Isolation and High-throughput Sequencing Library Construction for Herbarium Specimens

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with a number of challenges including sample preservation quality, degraded DNA, and destructive sampling of rare specimens. In order to more effectively use herbarium material in large sequencing projects, a dependable and scalable method of DNA isolation and library preparation is needed. This paper demonstrates a robust, beginning-to-end protocol for DNA isolation and high-throughput library construction from herbarium specimens that does not require modification for individual samples. This protocol is tailored for low quality dried plant material and takes advantage of existing methods by optimizing tissue grinding, modifying library size selection, and introducing an optional reamplification step for low yield libraries. Reamplification of low yield DNA libraries can rescue samples derived from irreplaceable and potentially valuable herbarium specimens, negating the need for additional destructive sampling and without introducing discernible sequencing bias for common phylogenetic applications. The protocol has been tested on hundreds of grass species, but is expected to be adaptable for use in other plant lineages after verification. This protocol can be limited by extremely degraded DNA, where fragments do not exist in the desired size range, and by secondary metabolites present in some plant material that inhibit clean DNA isolation. Overall, this protocol introduces a fast and comprehensive method that allows for DNA isolation and library preparation of 24 samples in less than 13 h, with only 8 h of active hands-on time with minimal modifications.

This article demonstrates a detailed protocol for DNA isolation and high-throughput sequencing library construction from herbarium material including rescue of exceptionally poor-quality DNA.

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

Herbaria are an invaluable source of plant material that can be used in a variety of biological studies. The use of herbarium specimens is associated with ...

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our entire society's ability to treat systemic infections. In the United States alone, antibiotic-resistant infections kill approximately 23,000 people a year and cost around 20 billion USD in additional healthcare. One approach to combat antimicrobial resistance is combination therapy, which is particularly useful in the critical early stage of infection, before the infecting organism and its drug resistance profile have been identified. Many antimicrobial treatments use combination therapies. However, most of these combinations are additive, meaning that the combined efficacy is the same as the sum of the individual antibiotic efficacy. Some combination therapies are synergistic: the combined efficacy is much greater than additive. Synergistic combinations are particularly useful because they can inhibit the growth of antimicrobial drug resistant strains. However, these combinations are rare and difficult to identify. This is due to the sheer number of molecules needed to be tested in a pairwise manner: a library of 1,000 molecules has 1 million potential combinations. Thus, efforts have been made to predict molecules for synergy. This article describes our high-throughput method for predicting synergistic small molecule pairs known as the Overlap2 Method (O2M). O2M uses patterns from chemical-genetic datasets to identify mutants that are hypersensitive to each molecule in a synergistic pair but not to other molecules. The Brown lab exploits this growth difference by performing a high-throughput screen for molecules that inhibit the growth of mutant but not wild-type cells. The lab's work previously identified molecules that synergize with the antibiotic trimethoprim and the antifungal drug fluconazole using this strategy. Here, the authors present a method to screen for novel synergistic combinations, which can be altered for multiple microorganisms.

High-throughput Identification of Synergistic Drug Combinations by the Overlap2 Method

Synergistic drug combinations are difficult and time-consuming to identify empirically. Here, we describe a method for identifying and validating synergistic small molecules.

通过重叠2方法高通量识别协同药物组合

Although antimicrobial drugs have dramatically increased the lifespan and quality of life in the 20th century, antimicrobial resistance threatens our ...

Manipulation of Ploidy in Caenorhabditis elegans

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has been associated with both the progression of cancer and the development of drug resistance. Until now, it has not been feasible to easily manipulate the ploidy of a multicellular animal without generating mostly sterile progeny. Presented here is a simple and rapid protocol for generating tetraploid Caenorhabditis elegans animals from any diploid strain. This method allows the user to create a bias in chromosome segregation during meiosis, ultimately increasing ploidy in C. elegans. This strategy relies on the transient reduction of expression of the rec-8 gene to generate diploid gametes. A rec-8 mutant produces diploid gametes that can potentially produce tetraploids upon fertilization. This tractable scheme has been used to generate tetraploid strains carrying mutations and chromosome rearrangements to gain insight into chromosomal dynamics and interactions during pairing and synapsis in meiosis. This method is efficient for generating stable tetraploid strains without genetic markers, can be applied to any diploid strain, and can be used to derive triploid C. elegans. This straightforward method is useful for investigating other fundamental biological questions relevant to genome instability, gene dosage, biological scaling, extracellular signaling, adaptation to stress, development of resistance to drugs, and mechanisms of speciation.

Manipulation of Ploidy in Caenorhabditis elegans

This method allows for the generation of tetraploid and triploid Caenorhabditis nematodes from any diploid strain. Polyploid strains generated by this method have been used to study chromosome interactions in meiotic prophase, and this method is useful for examining important basic questions in cell, developmental, evolutionary, and cancer biology.

在秀丽线虫中对倍体的操控

Mechanisms that involve whole genome polyploidy play important roles in development and evolution; also, an abnormal generation of tetraploid cells has ...

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture (4C-seq)

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and effect sizes of regulatory elements to a target gene. Some progress has been made with the bioinformatic prediction of the existence and function of proximal epigenetic modifications associated with activated gene expression using conserved transcription factor binding sites. Chromatin conformation capture studies have revolutionized our ability to discover physical chromatin contacts between sequences and even within an entire genome. Circular chromatin conformation capture coupled with next-generation sequencing (4C-seq), in particular, is designed to discover all possible physical chromatin interactions for a given sequence of interest (viewpoint), such as a target gene or a regulatory enhancer. Current 4C-seq strategies directly sequence from within the viewpoint but require numerous and diverse viewpoints to be simultaneously sequenced to avoid the technical challenges of uniform base calling (imaging) with next generation sequencing platforms. This volume of experiments may not be practical for many laboratories. Here, we report a modified approach to the 4C-seq protocol that incorporates both an additional restriction enzyme digest and qPCR-based amplification steps that are designed to facilitate a greater capture of diverse sequence reads and mitigate the potential for PCR bias, respectively. Our modified 4C method is amenable to the standard molecular biology lab for assessing chromatin architecture.

High-throughput Identification of Gene Regulatory Sequences Using Next-generation Sequencing of Circular Chromosome Conformation Capture 4C-seq

The identification of physical interactions between genes and regulatory elements is challenging but has been facilitated by chromosome conformation capture methods. This modification to the 4C-seq protocol mitigates PCR bias by minimizing over-amplification of PCR templates and maximizes the mappability of reads by incorporating an addition restriction enzyme digest step.

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

The identification of regulatory elements for a given target gene poses a significant technical challenge owing to the variability in the positioning and ...

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to perform and its ability to standardize the fitness of many strains to a wild-type organism. The technique is limited, however, by available phenotypic markers and the number of strains that can be assessed simultaneously, creating the need for a great number of replicate experiments. Concurrent with large numbers of experiments, the labor and material costs for quantifying bacteria based on phenotypic markers are not insignificant. To overcome these negative aspects while retaining the positive aspects, we have developed a molecular-based approach to directly quantify microorganisms after engineering genetic markers onto bacterial chromosomes. Unique, 25 base pair DNA barcodes were inserted at an innocuous locus on the chromosome of wild-type and mutant strains of Salmonella. In vitro competition experiments were performed using inocula consisting of pooled strains. Following the competition, the absolute numbers of each strain were quantified using digital PCR and the competitive indices for each strain were calculated from those values. Our data indicate that this approach to quantifying Salmonella is extremely sensitive, accurate, and precise for detecting both highly abundant (high fitness) and rare (low fitness) microorganisms. Additionally, this technique is easily adaptable to nearly any organism with chromosomes capable of modification, as well as to various experimental designs that require absolute quantification of microorganisms.

Digital PCR-based Competitive Index for High-throughput Analysis of Fitness in Salmonella

This molecular-based approach for determining bacterial fitness facilitates precise and accurate detection of microorganisms using unique genomic DNA barcodes that are quantified via digital PCR. The protocol describes calculating the competitive index for Salmonella strains; however, the technology is readily adaptable to protocols requiring absolute quantification of any genetically-malleable organism.

基于数字PCR的沙门氏菌健康高通量分析竞争指数

A competitive index is a common method used to assess bacterial fitness and/or virulence. The utility of this approach is exemplified by its ease to ...

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly sensitive. This protocol obtains precise measurements of gene expression from single worms or from bulk samples. The protocol presented here provides a novel adaptation of existing methods for complementary DNA (cDNA) preparation coupled to a nanofluidic RT-qPCR platform. The first part of this protocol, named &#8216;Worm-to-CT&#8217;, allows cDNA production directly from nematodes without the need for prior mRNA isolation. It increases experimental throughput by allowing the preparation of cDNA from 96 worms in 3.5 h. The second part of the protocol uses existing nanofluidic technology to run high-throughput RT-qPCR on the cDNA. This paper evaluates two different nanofluidic chips: the first runs 96 samples and 96 targets, resulting in 9,216 reactions in approximately 1.5 days of benchwork. The second chip type consists of six 12 x 12 arrays, resulting in 864 reactions. Here, the Worm-to-CT method is demonstrated by quantifying mRNA levels of genes encoding heat shock proteins from single worms and from bulk samples. Provided is an extensive list of primers designed to amplify processed RNA for the majority of coding genes within the C. elegans genome.

High-Throughput Quantitative RT-PCR in Single and Bulk C. elegans Samples Using Nanofluidic Technology

In this article a high-throughput protocol for fast and reliable determination of gene expression levels in single or bulk C. elegans samples is described. This protocol does not require RNA isolation and produces cDNA directly from samples. It can be used together with high-throughput multiplexed nanofluidic real-time qPCR platforms.

使用纳米流体技术的单体和散装 C. 电子样品 中的高通量定量 RT-PCR

This paper presents a high-throughput reverse transcription quantitative PCR (RT-qPCR) assay for Caenorhabditis elegans that is fast, robust, and highly ...

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors contribute to alterations in the normal aging process. These factors intersect in complex ways, as evidenced by the wealth of documented links identified and conserved in many organisms. Most of these studies focus on loss-of-function, null mutants that allow for rapid screening of many genes simultaneously. There is much less work that focuses on characterizing the role that overexpression of a gene in this process. In the present work, we present a straightforward methodology to identify and clone genes in the budding yeast, Saccharomyces cerevisiae, for study in suppression of the short-lived chronological lifespan phenotype seen in many genetic backgrounds. This protocol is designed to be accessible to researchers from a wide variety of backgrounds and at various academic stages. The SIR2 gene, which codes for a histone deacetylase, was selected for cloning in the pRS315 vector, as there have been conflicting reports on its effect on the chronological lifespan. SIR2 also plays a role in autophagy, which results when disrupted via the deletion of several genes, including the transcription factor ATG1. As a proof of principle, we clone the SIR2 gene to perform a suppressor screen on the shortened lifespan phenotype characteristic of the autophagy deficient atg1&#916; mutant and compare it to an otherwise isogenic, wild type genetic background.

A Suppressor Screen for the Characterization of Genetic Links Regulating Chronological Lifespan in Saccharomyces cerevisiae

Here is a protocol to identify genetic interactions through an increased copy number suppressor screen in Saccharomyces cerevisiae. This method allows researchers to identify, clone, and test suppressors in short-lived yeast mutants. We test the effect of the copy number increase of SIR2 on lifespan in an autophagy null mutant.

糖精中遗传链接调控时间寿命的抑制 器屏幕

Aging is the time dependent deterioration of an organism&#8217;s normal biological processes that increases the probability of death. Many genetic factors ...

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

摘要

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此视频中的章节

机器人平台的高通量原生质体分离和转型

核密度分析的天然染色质沉淀测序文库的生成

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

通过重叠²方法高通量识别协同药物组合

在秀丽线虫中对倍体的操控

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

基于数字PCR的沙门氏菌健康高通量分析竞争指数

使用纳米流体技术的单体和散装 C. 电子样品中的高通量定量 RT-PCR

糖精中遗传链接调控时间寿命的抑制器屏幕

复制集方法: 一种高通量方法定量测量秀丽线虫寿命

摘要

探索更多视频

此视频中的章节

机器人平台的高通量原生质体分离和转型

核密度分析的天然染色质沉淀测序文库的生成

线虫线虫线虫-一个多用途的体内模型研究宿主-微生物相互作用

植物标本标本的鲁棒 DNA 分离与高通量测序库建设

通过重叠2方法高通量识别协同药物组合

在秀丽线虫中对倍体的操控

利用下一代序列染色体构象捕获 (4 c 序列) 的高通量识别基因调控数列

基于数字PCR的沙门氏菌健康高通量分析竞争指数

使用纳米流体技术的单体和散装 C. 电子样品 中的高通量定量 RT-PCR

糖精中遗传链接调控时间寿命的抑制 器屏幕

通过重叠²方法高通量识别协同药物组合

使用纳米流体技术的单体和散装 C. 电子样品中的高通量定量 RT-PCR

糖精中遗传链接调控时间寿命的抑制器屏幕