우리는 분자 역학 시뮬레이션에서 생성 된 DNA 이중 나선의 좌표를 공유하기위한 지리산 Šponer에 감사합니다. 우리는 또한 이러한 구조를 다운로드에 도움 나다 Spackova을 인정합니다. USPHS 연구 보조금을 통해이 작업을 지원 GM34809 및 GM096889은 기꺼이 인정 받고 있습니다.

<ol>
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관심 없음 충돌 선언하지 않습니다.

이 문서에 제시된 프로토콜의 집합은 프로그램의 3DNA 제품군의 기능에 터치합니다. 도구 등 같은 페어링이 발생하는 이차 구조 컨텍스트를 결정하는 나선형 조각의 공간적 배치를 정량화하기 위해, 체인 백본을 따라 기지의 중복을 측정하기 위해, 비정규 기본 쌍을 식별하는 구조를 RNA에 적용 할 수 rebuild 명령은 사용자가 1 그림 삽입에 표시된 것과 기지와 기지 쌍의 간단하고 유익?...

DNA, RNA, 단백질, 마약, 그리고 다른 리간드와의 착물의 입체 구조를 이해하는 것은 그들의 다양한 생물학적 기능을 해독 매우 중요하며, 치료의 합리적인 디자인을 허용합니다. 분석 (모양과 상호 작용 패턴을 추출하는), 모델링 (에너지 론과 분자 역학을 평가하기 위해), 및 시각화 : 같은 구조의 탐사 별도의 세 가지 아직 밀접하게 관련 구성 요소를 수반한다. 구조 분석 및 모델 구축은 동전 본질적으로 두 가지 측면, 그리고 시각화 둘 다 보완한다. 컴퓨터 프로그램의 3DNA 제품군은 세 가지 차원 핵산 구조를 분석 구성 및 시각화하는 기능을 점점 더 인기를 구조 생물 정보학 툴킷입니다. 이전 출판물 조리법 선택한 작업 2를 수행하기 위해 제공되는 소프트웨어 1의 기능을 설명, 웹 기반 인터페이스를 도입소프트웨어 3의 인기있는 기능으로, 구조 기능을 제공 데이터베이스를 사용하여 수집 3DNA 4, 5는 DNA와 RNA의 구조 6, 7 모두 분석 소프트웨어의 유틸리티를 설명합니다. 이 문서의 목적은 DNA와 최첨단 전산 도구를 사용하여 RNA 공간 조직을 조사하기 위해 이익 및 / 또는 필요로하는 실험실 과학자와 다른 사람에게 3DNA 소프트웨어 키트를 가지고있다. 여기에 제시 프로토콜은 단계별 지침 (I) 맥 OS X 시스템에서 소프트웨어를 다운로드하고 설치하려면 (II-III) 구성 기본 쌍 단계의 수준에서 DNA 구조를 분석하고 수정하는 방법 (포함 IV-V) 분석 및 관련 DNA 구조의 집합을 정렬하고 (VI) 사용자 친화적 w3DNA 웹 인터페이스 단백질 장식 DNA 사슬의 모델을 구성 할 수 있습니다. 소프트웨어는 각각의 구조뿐만 아니라 큰 X-선 결정학 방법을 사용하여 해결할 분석하는 기능을 가지고 있습니다핵 자기 공명 (NMR) 방법으로 측정 또는 컴퓨터 시뮬레이션 기술에 의해 생성 된 구조의 앙상블. 구조 (I) 보렐리 아 burgdorferi 8 (인간 9, 10에서 라임 병을 일으키는 원인이되는 진드기 매개 박테리아), (ⅱ)에서 HBB 단백질에 바인딩 DNA의 고해상도 결정 구조를 포함 여기에 순차적으로 두 개의 큰 세트를 검토 D 4,500 스냅 샷 (GGCAAAATTTTGCC 2)와 D (CCGTTTTAAAACGG 2) 계산시 100 psec로 단위에서 수집하고, O3의 DNA 연산자의 NMR 기반 구조 (III) 작은 앙상블 - 분자 시뮬레이션을 11로 생산 관련 DNA 분자 대장균 락 억제 단백질 (12)의 투구에 바인딩됩니다. 아래의 지침은 이러한 구조의 각각뿐만 아니라 방법 3DNA (이 파일의 복사본을 찾을 수 있습니다 사용하는 연결된 원자 좌표 파일에 액세스하는 방법에 대한 정보를 포함에서 3DNA 포럼 <a href="http://forum.x3dna.org/jove" target="_blank">http://forum.x3dna.org/jove</a> ) 이러한 구조를 검사하고 수정합니다.

analyzing and building nucleic acid structures with 3dna

3DNA 소프트웨어 패키지, 분석 구축, 3 차원 핵산 구조를 시각화하는 기능을 갖춘 인기있는 다양한 생물 정보학 도구입니다. 이 문서에서는 각각의 구조 및 관련 구조의 앙상블에 모두 적용 3DNA에서 사용할 수있는 새로운 대중적인 기능의 하위 집합에 대한 상세한 프로토콜을 제공합니다. 프로토콜 1은 소프트웨어를 다운로드하고 설치하는 데 필요한 지침의 집합을 나열합니다. 이 기본 쌍의 할당 및 구조를 설명 강체 매개 변수의 결정을 포함하는 핵산 구조의 분석의 프로토콜 2에 따르고, 프로토콜 3, 원자의 재건에 대해 설명합니다 의 강체 매개 변수와 구조의 모델입니다. 3DNA, 버전 2.1의 가장 최근 버전은 핵 자기 공명 (NMR) 측정 및 분자 동적 (MD에서 추론 것과 같은 구조의 앙상블의 분석 및 조작을위한 새로운 기능을 가지고 있습니다) 시뮬레이션, 이러한 기능은 프로토콜 4와 5에 제시되어있다. 3DNA 독립형 소프트웨어 패키지 외에도,에 위치한 w3DNA 웹 서버, <a href="http://w3dna.rutgers.edu" target="_blank">http://w3dna.rutgers.edu는</a> , 소프트웨어의 선택 기능과 사용자 친화적 인 인터페이스를 제공합니다. 프로토콜 6 사용자가 지정한 위치에 바인딩 단백질과 장식 긴 DNA 분자의 모델을 구축하는 사이트의 새로운 기능을 보여줍니다.

3DNA 소프트웨어 도구는 정기적으로 핵산의 구조를 분석하는 데 사용됩니다. 예를 들어, 기본 쌍의 신원과 DNA와 RNA 구조의 이중 나선형 조각의 염기 배열을 특징 강체 매개 변수가 자동 핵산 데이터베이스 22의 세계적인 저장소의 각 새 항목에 대해 계산 및 저장 핵산 구조 정보를 제공합니다. 프로토콜 2 결정 강체 매개 변수의 값은 쉽게에서 발견, 대형 긍정적 인 롤 각도 (64.95 °, 60.93 °)로...

Watch this Scientific Journal Video about Analyzing and Building Nucleic Acid Structures with 3DNA at JoVE.com

Analyzing and Building Nucleic Acid Structures with 3DNA

3DNA 소프트웨어 패키지, 분석 구축, 3 차원 핵산 구조를 시각화하는 기능을 갖춘 인기있는 다양한 생물 정보학 도구입니다. 이 문서에서는 각각의 구조 및 관련 구조의 앙상블에 모두 적용 3DNA에서 사용할 수있는 새로운 대중적인 기능의 하위 집합에 대한 상세한 프로토콜을 제공합니다.

3DNA과 핵산의 구조를 분석하고 구축

The 3DNA software package is a popular and versatile bioinformatics tool with capabilities to analyze, construct, and visualize three-dimensional nucleic ...

analyzing-and-building-nucleic-acid-structures-with-3dna

Research

JoVE Journal

Biology

20.4K 조회수.  Rutgers - The State University of New Jersey. 3DNA 소프트웨어 패키지, 분석 구축, 3 차원 핵산 구조를 시각화하는 기능을 갖춘 인기있는 다양한 생물 정보학 도구입니다. 이 문서에서는 각각의 구조 및 관련 구조의 앙상블에 모두 적용 3DNA에서 사용할 수있는 새로운 대중적인 기능의 하위 집합에 대한 상세한 프로토콜을 제공합니다.

비디오: Analyzing and Building Nucleic Acid Structures with 3DNA

AC Electrokinetic Phenomena Generated by Microelectrode Structures

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, which is essential for Lab-on-a-Chip applications. AC electrokinetic phenomena use electric fields to generate forces that act on fluids or suspended particles (including those made of dielectric or biological material) and cause them to move in astonishing ways1, 2. Within a single channel, AC electrokinetics can accomplish many essential on-chip operations such as active micro-mixing, particle separation, particle positioning and micro-pattering. A single device may accomplish several of those operations by simply adjusting operating parameters such as frequency or amplitude of the applied voltage. Suitable electric fields can be readily created by micro-electrodes integrated into microchannels. It is clear from the tremendous growth in this field that AC electrokinetics will likely have a profound effect on healthcare diagnostics3-5, environmental monitoring6 and homeland security7.

In general, there are three AC Electrokinetic phenomena (AC electroosmosis, dielectrophoresis and AC electrothermal effect) each with unique dependencies on the operating parameters. A change in these operating parameters can cause one phenomena to become dominant over another, thus changing the particle or fluid behavior. 

It is difficult to predict the behavior of particles and fluids due to the complicated physics that underlie AC electrokinetics. It is the goal of this publication to explain the physics and elucidate particle and fluid behavior. Our analysis also covers how to fabricate the electrode structures that generate them, and how to interpret a wide number of experimental observations using several popular device designs. This video article will help scientists and engineers understand these phenomena and may encourage them to start using AC Electrokinetics in their research.

Manipulating fluids and suspended particles in the micro- and nano-scale is becoming more of a reality as enabling technologies, like AC electrokinetics, continue to develop. Here, we discuss the physics behind AC electrokinetics, how to fabricate these devices and how to interpret the experimental observations.

Microelectrode 구조에 의해 발생 AC 계면 현상

The field of AC electrokinetics is rapidly growing due to its ability to perform dynamic fluid and particle manipulation on the micro- and nano-scale, ...

연구

생물학

Fluorescent Labeling of Drosophila Heart Structures

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of &alpha;-actinin in larval hearts. Additionally, we perform labeling of F-actin and &alpha;-actinin in myosin-GFP expressing adult flies and of &alpha;-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Fluorescent Labeling of Drosophila Heart Structures

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

의 형광 라벨링 Drosophila 심장 구조

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells ...

Electricity-Free, Sequential Nucleic Acid and Protein Isolation

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern for governments, industries and medical professionals worldwide. For example, EHECs, combined with Shigella, are responsible for the deaths of approximately 325,000 children each year and are particularly prevalent in the developing world where laboratory-based identification, common in the United States, is unavailable 1. The development and distribution of low cost, field-based, point-of-care tools to aid in the rapid identification and/or diagnosis of pathogens or disease markers could dramatically alter disease progression and patient prognosis. We have developed a tool to isolate nucleic acids and proteins from a sample by solid-phase extraction (SPE) without electricity or associated laboratory equipment 2. The isolated macromolecules can be used for diagnosis either in a forward lab or using field-based point-of-care platforms. Importantly, this method provides for the direct comparison of nucleic acid and protein data from an un-split sample, offering a confidence through corroboration of genomic and proteomic analysis.
Our isolation tool utilizes the industry standard for solid-phase nucleic acid isolation, the BOOM technology, which isolates nucleic acids from a chaotropic salt solution, usually guanidine isothiocyanate, through binding to silica-based particles or filters 3. CUBRC's proprietary solid-phase extraction chemistry is used to purify protein from chaotropic salt solutions, in this case, from the waste or flow-thru following nucleic acid isolation4.
By packaging well-characterized chemistries into a small, inexpensive and simple platform, we have generated a portable system for nucleic acid and protein extraction that can be performed under a variety of conditions. The isolated nucleic acids are stable and can be transported to a position where power is available for PCR amplification while the protein content can immediately be analyzed by hand held or other immunological-based assays. The rapid identification of disease markers in the field could significantly alter the patient's outcome by directing the proper course of treatment at an earlier stage of disease progression. The tool and method described are suitable for use with virtually any infectious agent and offer the user the redundancy of multi-macromolecule type analyses while simultaneously reducing their logistical burden.

A tool and chemistries are described to sequentially isolate nucleic acids followed by proteins from a sample without the need for electricity. The tool consists of a sorbent held within a transfer pipette while the isolation chemistries are based on solid-phase extraction principles. The isolated macromolecules can be analyzed by immuno-based and PCR-based assays.

전기 - 무료, 선형 핵산 및 단백질 분리

Traditional and emerging pathogens such as Enterohemorrhagic Escherichia coli (EHEC), Yersinia pestis, or prion-based diseases are of significant concern ...

Simultaneous Multicolor Imaging of Biological Structures with Fluorescence Photoactivation Localization Microscopy

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled single molecules within a sample with a spatial resolution of tens of nanometers. Using either photoactivatable (PAFP) or&#160;photoswitchable (PSFP) fluorescent proteins fused to proteins of interest, or organic dyes conjugated to antibodies or other molecules of interest, fluorescence photoactivation localization microscopy (FPALM) can simultaneously image multiple species of molecules within single cells. By using the following approach, populations of large numbers (thousands to hundreds of thousands) of individual molecules are imaged in single cells and localized with a precision of ~10-30 nm. Data obtained can be applied to understanding the nanoscale spatial distributions of multiple protein types within a cell. One primary advantage of this technique is the dramatic increase in spatial resolution: while diffraction limits resolution to ~200-250 nm in conventional light microscopy, FPALM can image length scales more than an order of magnitude smaller. As many biological hypotheses concern the spatial relationships among different biomolecules, the improved resolution of FPALM can provide insight into questions of cellular organization which have previously been inaccessible to conventional fluorescence microscopy. In addition to detailing the methods for sample preparation and data acquisition, we here describe the optical setup for FPALM. One additional consideration for researchers wishing to do super-resolution microscopy is cost: in-house setups are significantly cheaper than most commercially available imaging machines. Limitations of this technique include the need for optimizing the labeling of molecules of interest within cell samples, and the need for post-processing software to visualize results. We here describe the use of PAFP and PSFP expression to image two protein species in fixed cells. Extension of the technique to living cells is also described.

We demonstrate the use of fluorescence photo activation localization microscopy (FPALM) to simultaneously image multiple types of fluorescently labeled molecules&#160;within cells. The techniques described yield the localization of thousands to hundreds of thousands of individual fluorescent labeled proteins, with a precision of tens of nanometers&#160;within single cells.

Localization-based super resolution microscopy can be applied to obtain a spatial map (image) of the distribution of individual fluorescently labeled ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

타운 웨스턴 가장 빠른 : 클래식 웨스턴 블롯 분석에 현대 트위스트

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Methyltransferases과 공동 인자 유사체와 핵산과 단백질의 순서 특정 레이블

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Simple Bulk Readout of Digital Nucleic Acid Quantification Assays

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are still not routinely applied, due to the cost and specific equipment associated with commercially available methods. Here we present a simplified method for readout of digital droplet assays using a conventional real-time PCR instrument to measure bulk fluorescence of droplet-based digital assays.
We characterize the performance of the bulk readout assay using synthetic droplet mixtures and a droplet digital multiple displacement amplification (MDA) assay. Quantitative MDA particularly benefits from a digital reaction format, but our new method&#160;applies to any digital assay. For established digital assay protocols such as digital PCR, this method serves to speed up and simplify assay readout.
Our bulk readout methodology brings the advantages of partitioned assays without the need for specialized readout instrumentation. The principal limitations of the bulk readout methodology are reduced dynamic range compared with droplet-counting platforms and the need for a standard sample, although the requirements for this standard are less demanding than for a conventional real-time experiment. Quantitative whole genome amplification (WGA) is used to test for contaminants in WGA reactions and is the most sensitive way to detect the presence of DNA fragments with unknown sequences, giving the method great promise in diverse application areas including pharmaceutical quality control and astrobiology.

We describe an endpoint digital assay for quantifying nucleic acids with a simplified (analog) readout. We measure bulk fluorescence of droplet-based digital assays using a standard qPCR machine rather than specialized instrumentation and confirm our results by microscopy.

디지털 핵산 정량 분석​​ 실험의 단순 대량 판독

Digital assays are powerful methods that enable detection of rare cells and counting of individual nucleic acid molecules. However, digital assays are ...

Efficient Nucleic Acid Extraction and 16S rRNA Gene Sequencing for Bacterial Community Characterization

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing technologies have allowed for the rapid and efficient characterization of bacterial communities using 16S rRNA gene sequencing from a variety of sources. Although readily available tools for 16S rRNA sequence analysis have standardized computational workflows, sample processing for DNA extraction remains a continued source of variability across studies. Here we describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs. We also delineate downstream methods for 16S rRNA gene sequencing, including generation of sequencing libraries, data quality control, and sequence analysis. The workflow can accommodate multiple samples types, including stool and swabs collected from a variety of anatomical locations and host species. Additionally, recovered DNA and RNA can be separated and used for other applications, including whole genome sequencing or RNA-seq. The method described allows for a common processing approach for multiple sample types and accommodates downstream analysis of genomic, metagenomic and transcriptional information.

We describe an efficient, robust, and cost effective method for extracting nucleic acid from swabs for characterization of bacterial communities using 16S rRNA gene amplicon sequencing. The method allows for a common processing approach for multiple sample types and accommodates a number of downstream analytic processes.

세균 커뮤니티 특성화에 대한 효율적인 핵산 추출 및 16S rRNA의 유전자 시퀀싱

There is a growing appreciation for the role of microbial communities as critical modulators of human health and disease. High throughput sequencing ...

Filtration Isolation of Nucleic Acids:  A Simple and Rapid DNA Extraction Method

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad to extract cellular DNA from whole blood in less than 2 min. The blood specimen is treated with detergent, mixed briefly and applied by pipet to the separation membrane. The lysate wicks into the blotting pad due to capillary action, capturing the genomic DNA on the surface of the separation membrane. The extracted DNA is retained on the membrane during a simple wash step wherein PCR inhibitors are wicked into the absorbent blotting pad. The membrane containing the entrapped DNA is then added to the PCR reaction without further purification. This simple method does not require laboratory equipment and can be easily implemented with inexpensive laboratory supplies. Here we describe a protocol for highly sensitive detection and quantitation of HIV-1 proviral DNA from 100 &#181;l whole blood as a model for early infant diagnosis of HIV that could readily be adapted to other genetic targets.

We describe here a simple and rapid paper-based DNA extraction method of HIV proviral DNA from whole blood detected by quantitative PCR. This protocol can be extended for use in detecting other genetic markers or using alternative amplification methods.

핵산의 여과 분리 : 간단하고 신속한 DNA 추출 방법

FINA, filtration isolation of nucleic acids, is a novel extraction method which utilizes vertical filtration via a separation membrane and absorbent pad ...

Laser-Capture Microdissection RNA-Sequencing for Spatial and Temporal Tissue-Specific Gene Expression Analysis in Plants

The development of a complex multicellular organism is governed by distinct cell types that have different transcriptional profiles. To identify transcriptional regulatory networks that govern developmental processes it is necessary to measure the spatial and temporal gene expression profiles of these individual cell types. Therefore, insight into the spatio-temporal control of gene expression is essential to gain understanding of how biological and developmental processes are regulated. Here, we describe a laser-capture microdissection (LCM) method to isolate small number of cells from three barley embryo organs over a time-course during germination followed by transcript profiling. The method consists of tissue fixation, tissue processing, paraffin embedding, sectioning, LCM and RNA extraction followed by real-time PCR or RNA-seq. This method has enabled us to obtain spatial and temporal profiles of seed organ transcriptomes from varying numbers of cells (tens to hundreds), providing much greater tissue-specificity than typical bulk-tissue analyses. From these data we were able to define and compare transcriptional regulatory networks as well as predict candidate regulatory transcription factors for individual tissues. The method should be applicable to other plant tissues with minimal optimization.

Presented here is a protocol for laser-capture microdissection (LCM) of plant tissues. LCM is a microscopic technique for isolating areas of tissue in a contamination-free manner. The procedure includes tissue fixation, paraffin embedding, sectioning, LCM and RNA extraction. RNA is used in the downstream tissue-specific, temporally resolved analysis of transcriptomes.