Gostaríamos de agradecer L. Evans, B. Granger, D. Rissling, Z. Sim, A. Goodwin, K. Hayhurst e D. Kuch de assistência no campo. K. Galbreath gentilmente cedidas amostras de fígado pika do Coola Bella vale. Graças à A. Gonçalves da Silva e Larsen K. para discussões interessantes que contribuíram para a concepção deste método de amostragem não-invasiva genética. Este trabalho foi financiado pelas Ciências Naturais e Engenharia Council of Canada Discovery, e Okanagan UBC Bolsas individuais de investigação para março A suíça National Science Foundation Fellowship Doutorado PBSKP3_128523 apoiado PH. Este estudo foi realizado seguindo o protocolo de atendimento de animais da Universidade de British Columbia (Número do certificado: A07-0126).

1. Laço de cabelo Antes da criação de um laço, um local ideal deve ser determinada dentro do habitat pika ou inclinação do tálus. Isto inclui montes de feno, que são caches vegetação que recolhem os animais no final do verão, bem como fezes frescas encontradas em locais de latrina. Tiras de fita de embalagem (10-50cm de comprimento) são enrolados para prover uma superfície de 360 ​​° pegajosa e são organizados de uma forma como a web-envelope a entrada para o monte de feno pika (Fig. 1) ou site latrina. Dependendo da configuração das rochas, um pedaço de linha de pesca podem ser utilizados para apoiar a estrutura do laço do cabelo, mas esta muitas vezes não é necessário quando as entradas são muito pequenas (&lt;30 cm de diâmetro), uma descrição completa do uso de linha de pesca podem ser encontrados em Henry e Russello (2010) 3. Armadilhas cabelos são verificados o mais rápido possível, e amostras de cabelo depositados na fita (Fig. 2) são então coletados e rotulados. Uma vez transportado para o laboratório, os pêlos são removidos da fita adesiva com a pinça estéril e transferido para tubos criogênicos e armazenadas a -20 ° C até a manipulação posterior. Amostras de cabelo agrupados ao longo de um laço de cabelo são considerados como pertencentes a um único indivíduo, enquanto amostras de cluster são assumidos de forma independente de pertencer a indivíduos diferentes. Neste último caso, o cabelo é então colocado em tubos diferentes. Estas suposições podem depois ser testados por meio de impressões digitais de DNA e cálculos de probabilidade de identidade com base em dados de microssatélites genotípica. 2. Extração de DNA Desde que o cabelo pika é muito fino e contém uma lâmpada pequena raiz, temos mostrado previamente que um mínimo de 25 cabelos são obrigados a obter uma quantidade suficiente de DNA de boa qualidade para amplificação por PCR downstream 3. Utilizou-se o DNA de QI sistema (Promega, Madison, WI, EUA) e uma versão ligeiramente modificada de instruções do fabricante para o isolamento de DNA de amostras de cabelo. Em primeiro lugar, a amostra de cabelo é girada para baixo em uma centrífuga de micro-, a fim de evitar a perda de cabelo durante a abertura do tubo. Então a solução de incubação é preparada misturando 80 ml de solução de incubação com 10μl da TDT (1M) e 10μl de proteinase K (18ng/ml) para resultar em uma concentração final de 0,1 M DTT e 1.8ng/ml de Proteinase K. Incubação solução (100 l) é adicionado à amostra e incubados a 56 ° C por 1 hora. Nesse meio tempo, a solução de lise é preparada por adição de 1 ml de DTT (1M) para cada 100 mL de tampão de lise, resultando em uma concentração final de 0,01 M DTT. Solução de lise preparada (200 mL) é adicionado à amostra, juntamente com 7 mL de DNA de resina de QI, agitadas por 3 segundos em alta velocidade e incubadas em temperatura ambiente por 5 minutos. A amostra é então centrifugadas por 2 segundos e inserido no suporte magnético, onde a separação das esferas magnéticas da solução ocorre. A solução restante é então aspirado e descartado, tomando cuidado para não perturbar o pellet de resina magnética. A amostra é então tratada com 100 ml da solução de lise preparada, agitadas e voltou para o stand onde a separação magnética ocorre novamente. O tampão de lise é aspirado e descartado, e este passo é repetido três vezes usando um tampão de lavagem (100 l). A amostra é posteriormente deixada a secar no suporte magnético por 15 minutos. Depois de seco, 100 ml de tampão de eluição é adicionado à amostra e incubados a 65 ° C por 5 minutos. A amostra é agitadas e inserido no suporte magnético. A solução agora contendo o DNA eluída é então transferido para um tubo Eppendorf 1,5 ml e armazenadas a -20 ° C até manipulações mais. DNA de amostras de fígado também foi extraído usando o sistema de QI DNA e usado como um controle positivo para amplificações PCR. 3. Amplificação por PCR O DNA obtido a partir de nosso laço do cabelo não-invasivo e amostras de fígado foram então usados ​​para amplificar um conjunto de comumente utilizados marcadores moleculares (microssatélites, AFLP, mitocondrial citocromo B e ZFX / ZFY marcador sexagem). PCRs foram realizadas utilizando um ciclador térmico Veriti (Applied Biosystems, Foster City, CA, EUA) em um volume 25 mL contendo: 10 - 20 ng de DNA, 0,5 mM de cada primer, 10 mM Tris-HCl (pH 8,3), 50 mM KCl, 1,5 mM MgCl 2, 200 mM dNTPs, 10 μ albumina sérica bovina (BSA; New England Biolabs, Ipswich, MA, EUA) e 0,5 U AmpliTaq DNA polimerase Gold (Applied Biosystems). Parâmetros para o ciclismo loci microssatélites foram otimizados usando um programa de ciclismo touchdown (10 min a 95 ° C, 35 ciclos de 95 ° C por 30 s, 30 s de anelamento, e 45 s em 72 ° C, seguido por uma etapa final a 72 ° C por 10 min). A temperatura de anelamento diminuiu 1 ° C por ciclo de 60-55 ° C até atingir o sexto ciclo, altura em que os 29 ciclos restantes continuaram a 55 ° C. Parâmetros de ciclismo para os fragmentos foram mitocondrial, como descrito acima, mas consistia de 35 ciclos com um recozimentotemperatura de 50 ° C. Polimorfismo comprimento de fragmento amplificado foi realizada seguindo o protocolo de genomas de vertebrados descritos por Bonin 4. Por último, sexagem molecular foi realizada utilizando a PCR-RFLP de ZFX / ZFY loci com a enzima HinfI digestão restrição 5. Os produtos de PCR do microsatélite, AFLPs e seqüências do citocromo B foram executados em um analisador genético ABI 3130XL (Applied Biosystems), enquanto digerida ZFX / ZFX fragmentos foram executados ao lado de uma escada bp 100 (New England Biolabs) em um 3% agarose gel contendo 2,5% gel DNA SYBR Safe. mancha (Invitrogen, Carlsbad, CA, EUA) e visualizados através de um sistema de imagem pessoal RED gel (Alpha Innotech, San Leandro, CA, EUA) Microssatélites e AFLPs foram visualizados usando Genemapper v3.7 (Applied Biosystems) e do cromatograma DNA mitocondrial foi visualizado usando Sequencher v4.7 (Gene Código Corporation, Ann Harbour, Michigan, EUA). 4. Resultados representativos O DNA extraído de amostras de cabelo utilizando o nosso laço não-invasivos foram usados ​​para PCR amplificar vários tipos de marcadores moleculares. Como base para comparação, utilizando DNA extraído de amostras de fígado foi amplificado ao lado de nossas amostras de cabelo. Loci microssatélites nucleares foram amplificados com sucesso tanto para cabelos e amostras de fígado (Fig. 3). Embora a intensidade do sinal foi maior quando se utiliza a amostras de fígado, este não ter um efeito adverso sobre a pontuação genótipo (Fig. 3). Resultados semelhantes foram obtidos quando se utiliza AFLPs (Fig. 4), seqüências de DNA mitocondrial (Fig. 5), eo ZFX / ZFY marcador sexagem molecular (Fig. 6). <img src="/files/ftp_upload/2791/2791fig1.jpg" alt="Figura 1"/> Figura 1. Um exemplo de um laço de cabelo não-invasivo criado em uma pilha de feno. Tiras de fita adesiva enrolada clara de embalagem são organizados de forma web-like para delimitar a entrada para o monte de feno. Um pedaço de linha de pesca é aqui utilizado para fornecer suporte adicional para o laço do cabelo. <img src="/files/ftp_upload/2791/2791fig2.jpg" alt="Figura 2"/> Figura 2. Um laço de cabelo bem sucedida contendo um grande número de cabelo preso à fita de embalagem. <img src="/files/ftp_upload/2791/2791fig3.jpg" alt="Figura 3"/> Figura 3. Um cromatograma microssatélites representante nuclear (Ocp6) 8 como mostrado na Genemapper v3.7 (Applied Biosystems). O cromatograma top foi obtida pela amplificação do DNA do tecido do fígado, e é heterozigoto (354/358) neste locus. O cromatograma inferior representa um indivíduo diferente exibindo um genótipo heterozigoto (358/362) com base em DNA extraído de cabelo. Enquanto o sinal de cromatograma da amostra de cabelo tem uma intensidade menor do que a amostra de fígado, marcando o genótipo não é afectada por esta diferença de sinal. <img src="/files/ftp_upload/2791/2791fig4.jpg" alt="Figura 4"/> Figura 4. Um cromatograma AFLP representante (E31T32), como exibido na Genemapper v3.7 (Applied Biosystems). O cromatograma top foi obtida pela amplificação do DNA de tecido hepático, o cromatograma fundo representa o DNA extraído de cabelo. <img src="/files/ftp_upload/2791/2791fig5.jpg" alt="Figura 5"/> Figura 5. Cromatograma A seqüência representante do DNA mitocondrial (citocromo B), como exibido na Sequencher v4.7 (Gene Código Corporation). O cromatograma top foi obtida pela amplificação do DNA de tecido hepático, o cromatograma fundo representa o DNA extraído de cabelo. <img src="/files/ftp_upload/2791/2791fig6.jpg" alt="Figura 6"/> Figura 6. Um marcador de sexagem representante molecular (ZFX / ZFY) amplificado para fígado e amostras de cabelo. Esta abordagem se baseia na observação de que o cromossomo Y possui um sítio de restrição HinfI nesta região que o cromossomo X não tem. Assim, as fêmeas produzem dois co-migração de fragmentos de 432 pb, enquanto os machos produzem fragmentos de 432 bp, bp 261 e 171 bp.

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Não há conflitos de interesse declarados.

Amostragem genética não-invasiva tem se tornado uma alternativa atraente para os métodos tradicionais de captura ao vivo por várias razões. Primeiro, ao discretamente coleta de material biológico (por exemplo, fezes, pêlos, penas, saliva e muco) de populações selvagens, os pesquisadores podem estudar estas espécies, sem perturbar, manuseio, ou mesmo observá-los, reduzindo assim os riscos para animais e pesquisadores. Segundo, não-invasivo de amostragem genética permite que os biólogos para estudar populações de espécies raras e evasivo, uma tarefa que pode ser difícil com o mais tradicional live-trapping abordagens 6. E terceiro, NGS pode potencialmente aumentar tamanho da amostra, reduzindo perturbação para os animais, os esforços de amostragem, e os custos, contribuindo assim para minimizar os vieses nas estimativas de parâmetros populacionais 7. Este último ponto pode ser crucial quando se trata de espécies ameaçadas, já que estimativas tendenciosas dos parâmetros populacionais podem resultar em uma gestão inadequada. No artigo de vídeo atual nós descrevemos um método simples, romance, barato e não invasivo para amostra elusive pequenos mamíferos, utilizando o Pika-americana como um exemplo. Nós demonstramos que o DNA extraído de cabelo executa de forma semelhante ao DNA extraído de amostras de fígado em relação ao microssatélites, AFLP, mitocondrial e marcadores de sexagem, tornando este método de amostragem não-invasiva uma alternativa atraente para se viver de amostragem captura ou letal. No geral, nós antecipamos que este método será útil na fase de coleta de dados de estudos populacionais genéticos e comportamentais de espécies raras ou evasivo de pequenos mamíferos, enquanto minimizando o impacto sobre os organismos em estudo.

Laço do cabelo: <ul><li> Rolos de fita adesiva transparente (3M, St. Paul, MN, EUA) </li><li> Linha de pesca (£ 10) </li><li> Criogênicos tubo (2ml) </li><li> Pinças esterilizadas </li><li> Nitrogênio líquido Dewar (opcional) </li></ul> Extração de DNA: <ul><li> DNA IQ System (Cat. # DC6701; Promega) com o tecido e de cabelo Kit de extração (Cat. # DC6740; Promega). </li><li> Tecnologia MagneSphere Fique Separação Magnética (Cat. # Z5342; Promega) </li><li> Micro centrífuga </li><li> Forno de hibridação ou água do banho </li><li> 1,5 ml Eppendorf Tubos </li></ul>

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

Não invasivo de amostragem abordagens genéticas estão se tornando cada vez mais importante para estudar populações de animais selvagens. Uma série de estudos têm relatado o uso de técnicas de amostragem não-invasivo para investigar a genética da população e demografia das populações selvagens 1. Esta abordagem provou ser especialmente útil quando se trata de espécies raras ou evasivo 2. Embora alguns destes métodos foram desenvolvidos para cabelos amostra, fezes e outros materiais biológicos de carnívoros e mamíferos de tamanho médio, eles têm grande parte permaneceu testado em elusive pequenos mamíferos. Neste vídeo, apresentamos um romance, laço de cabelo barato e não-invasivos destinados a um mamífero elusive pequeno, o pika americano (Ochotona princeps). Descrevemos o general set-up do laço do cabelo, que consiste em tiras de fita adesiva organizado de uma forma web-como e colocados ao longo viajando rotas em habitat do pikas &quot;. Nós ilustrar a eficiência da armadilha para coletar uma grande quantidade de cabelo que pode ser coletado e trazido de volta para o laboratório. Nós, então, demonstrar o uso do QI DNA sistema (Promega) para isolar DNA e mostrar a utilidade deste método para ampliar comumente utilizados marcadores moleculares microssatélites, incluindo nucleares, polimorfismos fragmento amplificado comprimento (AFLPs), seqüências mitocondriais (800bp), bem como um marcador de sexagem molecular. No geral, demonstrar a utilidade deste laço do cabelo romance não-invasivos como uma técnica de amostragem para população biólogos dos animais selvagens. Nós antecipamos que essa abordagem será aplicável a uma variedade de pequenos mamíferos, abrindo áreas de investigação dentro das populações naturais, minimizando o impacto para os organismos estudo.

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

Nós apresentamos um método de amostragem não invasiva para coletar amostras de cabelo de forma eficiente a partir elusive pequenos mamíferos, como mostrado para o pika americano. Demonstramos a utilidade deste método por extração de DNA de amostras de cabelo e ampliando vários tipos de marcadores moleculares usados ​​em estudos de ecologia da vida selvagem e conservação.

A técnica de amostragem não invasiva cabelo obter DNA de alta qualidade a partir de Elusive Pequenos Mamíferos

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

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JoVE Journal

Biology

20.6K visualizações.  University of British Columbia, Okanagan Campus. Nós apresentamos um método de amostragem não invasiva para coletar amostras de cabelo de forma eficiente a partir elusive pequenos mamíferos, como mostrado para o pika americano. Demonstramos a utilidade deste método por extração de DNA de amostras de cabelo e ampliando vários tipos de marcadores moleculares usados ​​em estudos de ecologia da vida selvagem e conservação.

Vídeo: 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.

Obtenção de RNA de alta qualidade a partir de populações de células no tecido humano único post-mortem do cérebro

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

Biologia

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.

Não invasivo In Vivo Pequenos Animais MRI e MRS: Basic procedimentos experimentais

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.

Microarrays de DNA: Controle de Qualidade da amostra, Hibridação Array e Digitalização

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.

Extração de DNA de alto peso molecular de Esteiras Microbianas

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.

Geração de alta Chromatin Template DNA Immunoprecipitation Qualidade para seqüenciamento de alto rendimento (CHIP-seq)

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.

Um manual de tela pequena molécula Aproximando de alta capacidade Usando embriões Zebrafish

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

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Um método de extração de DNA híbrido para a Avaliação de comunidades bacterianas Qualitativa e Quantitativa de amostras de produção de aves de capoeira

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

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.

Uma nova técnica de Análise Quantitativa de perda de cabelo em ratos usando Análise Tons de Cinza

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.

A ferramenta Web para caminhos biológicos que gera alta qualidade de leitura mecânica

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.

Um protocolo da microdissecção da captação do laser que rende o RNA da alta qualidade dos ossos frescos-congelados do rato

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