Gostaríamos de agradecer as seguintes pessoas, sem cuja ajuda a coleção de tecidos de pinho não teria sido possível: Dr. Joe Nairn, Matt Bryman, Michael Bordeaux, Ujwal Bagal, Huizhe Jin, e Amanda Bouffier.

Parte 1: Colheita Árvore Depois que a árvore é selecionada e cortada, é importante trabalhar com o mesmo cuidado e tão rapidamente quanto possível. Como cada tipo de tecido diferente é colhida, devem ser colocados imediatamente em suas próprias embarcações nitrogênio líquido para evitar a contaminação cruzada de material da amostra. Este protocolo de isolamento do RNA é escalável. <ol><li> Cortar os parafusos em comprimentos de cerca de 0,5-0,75 m e com uma pontuação motosserra a casca em duas ou três lugares seguintes do eixo longitudinal do parafuso. </li><li> Utilizando um cinzel de madeira, o trabalho da borda da região marcou até a casca começa a se separar da madeira e continuar a trabalhar o cinzel enquanto puxa para trás na seção de casca até que ela se separa da haste. </li><li> Xilema secundário é colhida com uma navalha reta de ponta, de lado único raspando ao longo do eixo longitudinal do parafuso que tem sido descascada. Usar luvas de látex para minimizar a contaminação do tecido. </li><li> Floema, que está localizado na parte interna das seções de casca, é prontamente colhidas pelo manual peeling. </li><li> Madeira de reação é coletado do lado de membros após marcando-as com tinta spray para indicar sua orientação original com respeito no chão. </li><li> Membros são cortados em parafusos de m 1-2 e marcou em lados opostos ao longo do eixo longitudinal de acordo com as localizações aproximadas dos dois tipos de madeira de reação: madeira oposto (no lado superior do membro) e madeira de compressão (no fundo lado do membro). A casca é separada do membro, como descrito acima, exceto que apenas a casca que cobre cada tipo de madeira de reação é removida de uma vez. Xilema secundário ou floema é, então, coletadas como descrito acima e colocada em nitrogênio líquido. </li><li> Outras amostras, como agulhas, brotos e cones jovens, podem ser colhidas à mão ou usando tesouras de poda, conforme necessário. </li><li> Amostras coletadas congeladas em nitrogênio líquido deve ser colocado em sacos ou recipientes adequados marcados e embalados em gelo seco para a expedição para o laboratório. </li></ol> Parte 2: Processamento Freezer Moinho de Amostras <ol><li> Encha o reservatório de moinho Spex 6850 freezer com nitrogênio líquido e coloque a barra de impacto em um dos frascos de moagem e preencher com nitrogênio líquido para chill-pre. Deitar fora nitrogênio líquido e adicione a amostra para o frasco. </li><li> Certifique-se de que nenhum excesso de nitrogênio líquido permanece na câmara de moagem e que o gás nitrogênio em excesso é completamente liberada antes da tampa está sentado para selar a câmara. </li><li> Insira o frasco de moagem para a transportadora e fechar. </li><li> Amostras woody moinho para dois ciclos de 1 minuto / ciclo à definição da taxa de 14. </li><li> Use uma espátula de nitrogênio líquido refrigerado para retirar a amostra moída (farinha de madeira) em um copo que foi pré-refrigerados e contém uma pequena quantidade de nitrogênio líquido. </li><li> Despeje a polpa de nitrogênio / amostra de líquido em um recipiente de armazenamento e coloque a -80 ° C até ser necessário. </li></ol> Parte 3: RNA Isolation, Dia 1 <ol><li> Coloque 20 mL de RNA do buffer de isolamento em um tubo Falcon 50 ml nivelado e acrescentar 400 brevemente β-mercaptoetanol vortex, mL, então o calor em banho-maria a 60 ° C. </li><li> Adicionar 4 g de tecido congelado a cada tubo. Tampa e agitar vigorosamente à mão seguido por vórtex por 10 segundos. </li><li> Adicionar um volume igual de clorofórmio para o tubo e inverter suavemente para misturar. Colocar o tubo em uma roda giratória e permitir end-over-end mistura por 5 minutos. </li><li> Decantar a mistura em um tubo de 50 mL de Ridge Oak e centrifugar a 12.000 (17.200 xg) rpm em um SS34 rotor de ângulo fixo por 5 minutos. </li><li> Transferir a fase aquosa para um novo tubo Oak Ridge e adicionar uma metade do volume de clorofórmio. </li><li> Vortex por 1 minuto e continue misturando em uma roda giratória por 5 minutos. Centrifugar a 12.000 rpm (17,200 xg) em um ângulo de rotor SS34 fixo por 5 minutos. </li><li> Transferir a fase aquosa para novo tubo de Oak Ridge e adicionar uma metade do volume de clorofórmio. Vortex por 1 minuto. Centrifugar a 12.000 rpm (17,200 xg) em um ângulo de rotor SS34 fixo por 10 minutos. </li><li> Transferir a fase aquosa para um tubo de 50 mL de polipropileno de alta velocidade. Centrifugar a 10.000 rpm (11,900 xg) em um ângulo de rotor SS34 fixo por 20 minutos. </li><li> Decantar o sobrenadante do tubo Falcon em 50 mL e adicionar um quarto volume 10 M solução de LiCl para sobrenadante. Brevemente vortex e colocar a 4 ° C para a precipitação durante a noite. </li></ol> Parte 4: RNA Isolation, Dia 2 <ol><li> Buffer de calor SSTE em um banho de água 65 ° C antes de iniciar. </li><li> Verter a amostra LiCl-precipitado em um tubo de polipropileno eo pellet de RNA por centrifugação a 11.000 rpm em um ângulo de rotor SS34 fixo (14.450 xg) por 30 minutos a 4 ° C. </li><li> Após a centrifugação,Deitar fora e descartar o líquido, e inverter os tubos em Kimwipes por aproximadamente 1 minuto. Pipeta de desconto em qualquer sobrenadante restante. </li><li> Ressuspender cada pellet em 800 mL de SSTE aquecido utilizando uma pipeta para misturar as amostras. Transferir a amostra em suspensão para tubos de 2 mL Ambion RNAse-free microcentrífuga. </li><li> Amostras de calor a 65 ° C por 2 minutos seguido por vórtex vigoroso para garantir a ressuspensão completa. </li><li> Adicionar um volume igual de fenol-clorofórmio, PH8, a cada tubo de amostra. Vortex cada amostra por 30 segundos e em seguida, girar em uma microcentrífuga a 14.000 rpm por 3 minutos </li><li> Transferir a fase aquosa para um novo Ambion 2mL tubos de microcentrífuga RNAse-free, e adicionar um volume igual de clorofórmio. Vortex cada amostra por 30 segundos e girar na microcentrífuga a 14.000 rpm por 3 minutos. </li><li> Transferência de fase aquosa para tubos Phase Lock-Gel que tenham sido previamente preparado por microfuging a 11.000 rpm por 2 minutos. Adicionar uma metade do volume de clorofórmio e gentilmente pipeta para misturar. </li><li> Centrifugar a 11.000 rpm por 5 minutos e transferir a fase aquosa para novo 2mL Ambion tubos de microcentrífuga RNAse-free. </li></ol> Parte 5: Precipitação e RNA Wash Etanol <ol><li> Adicionar 50 mL de acetato de sódio 3,0 M, pH 4,8, e 1 mL de etanol a 100% para a fase aquosa. Vortex e precipitado em -80 ° C por 30 minutos ou durante a noite a -20 ° C. </li><li> Amostras de microcentrífuga a 14.000 rpm por 30 minutos a 4 ° C, e aspirar cuidadosamente fora do sobrenadante. Não perturbar o pellet. </li><li> Lavar os pellets de RNA com 1 mL de etanol -20 ° C a 70% (v / v). Microcentrífuga a 14.000 rpm por 1 minuto e aspirado de fora o etanol. </li><li> Repita o passo 5.3. </li><li> Destampe a tubos e ar seco pellets no banco por 3-5 minutos. </li></ol> Parte 6: ressuspensão final e Quantificação <ol><li> Ressuspender cada pellet em 100 de água DEPC tratados mL. Misturar em vortex e incubar os tubos a 65 ° C por 2 minutos antes de colocar no gelo. Repita se necessário para garantir a ressuspensão da amostra. </li><li> Piscina como amostras, se desejar e fazer 50 mL de uma diluição de 1:10 em 10 mM Tris, pH 7,5, para a quantificação espectrofotométrica. Razão de absorvância para 260/280 e 260/230 são normalmente maiores do que 2,1 e 2,2, respectivamente. Isolamento do RNA rendimento varia de 60-120μg / g de tecido congelado. </li><li> Analisar amostras executando 1,5-2,5 mg RNA em um gel de agarose 1% em tampão TAE. Para mais amostras críticas, analisar o RNA usando um Agilent 2100 Bioanalyzer conforme instruções do fabricante. </li></ol> Resultados representativos: Rendimentos de isolamento de RNA de diferentes tecidos de coníferas faixa 60-120 mg / g tecido congelado com amostras woody normalmente produzindo em torno de 70-80g / g de tecido congelado. Índices de diagnóstico de 260/280 e 260/230 são normalmente maiores do que 2.1, e são bons indicadores de que a amostra de RNA é livre de qualquer contaminação ou fenólicos significativa de carboidratos, respectivamente. Amostras analisadas por eletroforese em gel de agarose corado pela EtBr deve mostrar três bandas distintas para 25S, 18S e RNA 5S (Figura 1). Determinar a estequiometria da 28S a 18S em gel de agarose é um tanto subjetiva, e fatores como a execução de condições para o gel, a coloração, ea carga de amostra podem ter um efeito. Em geral, o 28S: 18S relação parecerá ser&gt; 1,5. No entanto, algumas amostras de coníferas têm índices mais baixos. Por exemplo, após análise Agilent 2100 vimos vários casos de amostras de coníferas de diferentes tipos de tecidos, onde a estequiometria está mais perto de 1,2:1. Assim, uma aparente baixa 28S: 18S estequiometria por eletroforese em gel de agarose não indica necessariamente uma amostra de má qualidade. Folha, atirar, e amostras de cone, muitas vezes, têm adicional bandas distintas presente devido à chloroplastic RNAs ribossomais. EtBr manchada de materiais que não migram a partir da amostra bem ou bandas de alta massa molecular (&gt; 80-10 kb) geralmente indicam a contaminação do DNA genômico (Figura 2). Baixo peso molecular manchas massa nas proximidades ou abaixo da banda de 5S e coloração banda reduzida ribossomal 18S ou um aparente&gt; estequiometria 28S é um bom indicador de que o RNA degradação ocorreu (Figura 3). Na análise electrofograma Agilent 2100, a linha de base deve ser baixa e relativamente suave, com grandes picos correspondentes a 18S e 28S ribossomal ter RNAs 28S: 18S proporções que variam 1,2-2,0. A Agilent Número Integrity RNA (RIN) valor pode ser um melhor indicador de qualidade de RNA e deve ser pelo menos 7 ou superior para RNA que está a ser utilizado para a preparação de alvos microarray. A Figura 4 mostra uma típica Agilent 2100 electroferograma de RNA xilema com uma 28S: 18S proporção igual a 1,4 e um valor RIN de 8,6 enquanto a Figura 5 mostra um xilema pobres RNA prep ter uma linha de base muito alta e degradação perceptível como revelado pela 28S: 18S ratio igual a 0,65 e um valor de 3,4 RIN. <img src="/files/ftp_upload/1751/1751fig1.jpg" alt="Figura 1" /> Figura 1. Gel Agarose análise de RNA de alta qualidade de pinho. Raia 1, NEB 1 kb padrão, Lane 2 mostra um típico isolamento do RNA total de xilema secundário com pinheiros loblolly característica 28S ribossomal, 18S, 5S e 28S bandas e aparente: a relação 18S&gt; 1. <img src="/files/ftp_upload/1751/1751fig2.jpg" alt="Figura 2" /> Figura 2. Gel Agarose análise da amostra de RNA de pinheiros contaminadas com DNA genômico. Raia 1, NEB 1 kb padrão, Lane 2, amostra de RNA xilema mostrando a contaminação do DNA genômico. <img src="/files/ftp_upload/1751/1751fig3.jpg" alt="Figura 3" /> Figura 3. Gel Agarose análise de amostra de pinheiro mostrando RNA degradação e contaminação com DNA genômico. Raia 1, NEB 1 kb padrão, Lane2, amostra xilema RNA mostrando a contaminação e degradação do DNA genômico RNA grave. <img src="/files/ftp_upload/1751/1751fig4.jpg" alt="Figura 4" /> Figura 4. Agilent electrofograma 2100 de RNA total xilema isolados utilizando o método descrito no xilema mudas de pinus. 28S: 18S é igual a relação de 1,4, valor igual a 8,6 RIN <img src="/files/ftp_upload/1751/1751fig5.jpg" alt="Figura 5" /> Figura 5. Agilent 2100 electroferograma de RNA total de ponta apical mostrando severa degradação e contaminação genômica. 28S: 18S é igual a relação de 0,65, valor igual a 3,4 RIN

<ol>
	<li>Chang, S., Puryear, J., Cairney, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+simple+and+efficient+method+for+extracting+RNA+from+pine+trees.">A simple and efficient method for extracting RNA from pine trees.</a> Plant Molecular Biology Reporter. 11 (2), 113-116 (1993).</li><li>Lorenz, W. W., Yu, Y. S., Sim&#369;es, M., Dean, J. F. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+the+Loblolly+Pine+PtGen2+cDNA+Microarray.">Processing the Loblolly Pine PtGen2 cDNA Microarray.</a> Journal of Visualized Experiments. 25, (2009).</li></ol>

Obtenção de RNA de alta qualidade a partir de espécies de coníferas pode ser uma tarefa difícil, dado os altos níveis de compostos fenólicos e polissacarídeos encontrados em tecidos lenhosos. Começando com o protocolo desenvolvido por Chang et al. (1), descobrimos que a extração de mais rigoroso e limpar passos conduzem ao isolamento consistente de muito alta qualidade RNA total do woody woody vários e não-tecidos amostrados de uma grande variedade de espécies de coníferas. Este vídeo tem demonstrado nã...

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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<tr>
<td>RNA Isolation Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>2% CTAB (hexadecyltrimethylammonium bromide), 2% PVP (polyvinyl pyrrolidinone; Mw 30-40,000), 100mM Tris-HCl (pH8.0), 25mM EDTA, 2.0M NaCl, 0.5g/L Spermidine</td>
</tr>
<tr>
<td>Chloroform</td>
<td>&nbsp;</td>
<td>Fisher Scientific</td>
<td>C298-4</td>
<td>&nbsp;</td>
<tr>
<td>Phenol, Ultrapure</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>15509-037</td>
<td>&nbsp;</td>
<tr>
<td>10M LiCl</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
</tr>
<tr>
<td>SSTE Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>1M NaCl, 0.5% SDS, 10mM Tris-HCl (pH8.0), 1mM EDTA (pH8.0)</td>
</tr>
<tr>
<td>Sodium acetate (NaOAc) 3M pH4.8</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Made with DEPC-treated water</td>
</tr>
<tr>
<td>Phenol-chloroform (pH8.0)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>120mL phenol, 160mL chloroform titrated with multiple changes of 0.5M Tris-Cl pH 8.0</td>
</tr>
<tr>
<td>Oak Ridge High Speed Teflon Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-16B</td>
<td>&nbsp;</td>
<tr>
<td>Polypropylene 50mL High Speed Tubes</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#05-562-10K</td>
<td>&nbsp;</td>
<tr>
<td>BD Falcon,sterile, capped 50mL conical disposable tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>#21008-939</td>
<td>&nbsp;</td>
<tr>
<td>Phase Lock Gel Heavy, 2mL</td>
<td>&nbsp;</td>
<td>Thermo-Fisher</td>
<td>#2302830</td>
<td>&nbsp;</td>
<tr>
<td>Ambion RNase-free Microfuge Tubes, 2mL</td>
<td>&nbsp;</td>
<td>Ambion, Inc.</td>
<td>#AM12425</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

an improved method of rna isolation from loblolly pine (p. taeda l.) and other conifer species

Tecidos isolados de espécies de coníferas, particularmente aqueles que pertencem à família Pinaceae, como o pinus taeda (<em&gt; Pinus taeda</em&gt; L.), contêm altas concentrações de compostos fenólicos e polissacarídeos que interferem com a purificação de RNA. Isolamento de RNA de alta qualidade destas espécies requer procedimentos rigorosos de recolha de tecidos no campo eo emprego de um protocolo de isolamento de RNA composto de várias etapas de extração orgânica, a fim de isolar o RNA de qualidade suficiente para microarray e outras análises genômicas. O isolamento de RNA de alta qualidade a partir do campo de coleta de amostras de pinheiro pode ser um desafio, mas várias modificações para o tecido padrão e procedimentos de isolamento do RNA melhorar significativamente os resultados. A extensão da degradação geral RNA aumenta se as amostras não são devidamente recolhidos e transportados do campo, especialmente durante grande escala colheitas. Rendimentos totais RNA pode ser aumentada significativamente por amostras de pulverização em uma fábrica de nitrogênio líquido freezer antes de RNA de isolamento, especialmente quando as amostras provenientes de tecidos lenhosos. Isto é principalmente devido à presença de agentes oxidantes, tais como compostos fenólicos e polissacarídeos que estão presentes em níveis elevados em extratos de tecidos de espécies lenhosas mais coníferas. Se não forem removidos, esses contaminantes podem transitar levando a problemas como a degradação de RNA, que resultam em baixo rendimento e uma má qualidade da amostra de RNA. Transição de compostos fenólicos, bem como polissacarídeos, também pode reduzir ou mesmo eliminar completamente a atividade de transcriptase reversa ou polimerase outros comumente utilizados para a síntese de cDNA. Em particular, RNA destinado a ser usado como modelo para double-stranded síntese de cDNA na geração de bibliotecas de cDNA, single-stranded síntese de cDNA por PCR ou qPCR, ou para a síntese de materiais alvo microarray deve ser da mais alta qualidade se os pesquisadores esperam para obter melhores resultados. Técnicas de isolamento de RNA comumente empregados para muitas outras espécies de plantas são muitas vezes insuficientes na sua capacidade de remover estes contaminantes a partir de amostras de coníferas e, portanto, não produzem RNA total de amostras adequadas para manipulações a jusante. Neste vídeo podemos demonstrar os métodos para a coleta de campo de tecidos de coníferas, começando com a derrubada de uma árvore anos quarenta, para a colheita de floema, xilema secundário e xilema de madeira de reação. Nós também demonstramos um protocolo de isolamento de RNA que tem consistentemente rendeu RNA de alta qualidade para posterior manipulação enzimática.

Watch this Scientific Journal Video about An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species at JoVE.com

An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Muitos tecidos vegetais, incluindo floema e xilema do pinheiro (<em&gt; Pinus taeda</em&gt; L.), contêm altos níveis de compostos fenólicos e polissacarídeos que interferem com a purificação de RNA. Esta apresentação discute técnicas para a colheita de tecidos cultivados em campo e isolamento de RNA de qualidade suficiente para microarrays e outras análises genômicas.

Um método melhorado de isolamento de RNA Pine Loblolly ( P. taeda L.) e espécies de coníferas Outros

Tissues isolated from conifer species, particularly those belonging to the Pinaceae family, such as loblolly pine (Pinus taeda L.), contain high ...

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Research

JoVE Journal

Biology

An Improved Method of RNA Isolation from Loblolly Pine (P. taeda L.) and Other Conifer Species

13.7K visualizações.  University of Georgia (UGA). Muitos tecidos vegetais, incluindo floema e xilema do pinheiro ( Pinus taeda L.), contêm altos níveis de compostos fenólicos e polissacarídeos que interferem com a purificação de RNA. Esta apresentação discute técnicas para a colheita de tecidos cultivados em campo e isolamento de RNA de qualidade suficiente para microarrays e outras análises genômicas.

Vídeo: An Improved Method of RNA Isolation from Loblolly Pine P. taeda L. and Other Conifer Species

Processing the Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs. The array is produced in our laboratory for use by researchers studying gene expression in pine and other conifer species. PtGen2 was developed as a result of our gene discovery efforts in loblolly pine, and is comprised of sequences identified primarily from root tissues, but also from needle and stem.1,2 PtGen2 has been tested by hybridizing different Cy-dye labeled conifer target cDNAs, using both amplified and non-amplified indirect labeling methods, and also tested with a number of hybridization and washing conditions. This video focuses on the handling and processing of slides before and after pre-hybridization, as well as after hybridization, using some modifications to procedures developed previously.3,4 Also included, in text form only, are the protocols used for the generation, labeling and clean up of target cDNA s, as well as information on software used for downstream data processing. 
PtGen2 is printed with a proprietary print buffer that contains high concentrations of salt that can be difficult to remove completely. The slides are washed first in a warm SDS solution prior to pre-hybridization. After pre-hybridization, the slides are washed vigorously in several changes of water to complete removal of remaining salts. LifterSlips&trade; are then cleaned and positioned on the slides and labeled cDNA is carefully loaded onto the microarray by way of capillary action which provides for even distribution of the sample across the slide, and reduces the chance of bubble incorporation. Hybridization of targets to the array is done at 48&deg;C in high humidity conditions. After hybridization, a series of standard washes are done at 53&deg;C and room temperature for extended times. Processing PtGen2 slides using this technique reduces salt and SDS-derived artifacts often seen when the array is processed less rigorously. Hybridizing targets derived from several different conifer RNA sources, this processing protocol yielded fewer artifacts, reduced background, and provided better consistency among different experimental groups of arrays.

The cDNA microarray PtGen2 was developed for gene expression studies in loblolly pine, P. taeda, and other conifer species. Here, we show pre- and post-hybridization handling and washing techniques that can be used with this array to yield better consistency, reduced artifacts, and lower backgrounds.

Processamento do Loblolly Pine PtGen2 cDNA Microarray

PtGen2 is a 26,496 feature cDNA microarray containing amplified loblolly pine ESTs.  The array is produced in our laboratory for use by researchers ...

Biologia

RNA Isolation from Embryonic Zebrafish and cDNA Synthesis for Gene Expression Analysis

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead to disastrous results in downstream experimental applications.  It is therefore, of utmost importance to use solid techniques with numerous safeguards and quality control checks to ensure a superior sample.  Herein, we detail a protocol to isolate total RNA from whole zebrafish embryos using a commercially available chemical denaturant and subsequent cleanup to remove traces of DNA and impurities using a commercial RNA isolation kit. As RNA is relatively unstable and easily prone to cleavage by RNAses, most protocols assay gene expression using a cDNA product that is directly synthesized from an RNA template. We detail a procedure to convert RNA into the more stable cDNA product using a commercially available kit. Throughout these procedures there are numerous quality control checks to ensure that the sample is not degraded or contaminated.  The end product of these protocols is cDNA that is suitable for microarray analysis, RT-PCR or long-term storage.    

The isolation of high quality, intact RNA is an essential step in many laboratory protocols. Here, we demonstrate RNA extraction from whole zebrafish embryos and cDNA synthesis for subsequent application in various experimental procedures including gene expression microarray analysis.

Isolamento de RNA Síntese Zebrafish e cDNA embrionárias para Análise de Expressão Gênica

Many important and complex laboratory procedures require an input of high quality, intact RNA.  A degraded sample or the presence of impurities can lead ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Perfil de metiltransferases e Outros S-Adenosil- L Homocisteína-Proteínas de Ligação de captura por Espectrometria de Massa Compound (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Isolation and Purification of Kinesin from Drosophila Embryos

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a variety of neurodegenerative diseases, understanding microtubule based motor transport and its regulation will likely ultimately lead to improved therapeutic approaches. Kinesin-1 is a eukaryotic motor protein which moves in an anterograde (plus-end) direction along microtubules (MTs), powered by ATP hydrolysis. Here we report a detailed purification protocol to isolate active full length kinesin from Drosophila embryos, thus allowing the combination of Drosophila genetics with single-molecule biophysical studies. Starting with approximately 50 laying cups, with approximately 1000 females per cup, we carried out overnight collections. This provided approximately 10 ml of packed embryos. The embryos were bleach dechorionated (yielding approximately 9 grams of embryos), and then homogenized. After disruption, the homogenate was clarified using a low speed spin followed by a high speed centrifugation. The clarified supernatant was treated with GTP and taxol to polymerize MTs. Kinesin was immobilized on polymerized MTs by adding the ATP analog, 5'-adenylyl imidodiphosphate at room temperature. After kinesin binding, microtubules were sedimented via high speed centrifugation through a sucrose cushion. The microtubule pellet was then re-suspended, and this process was repeated. Finally, ATP was added to release the kinesin from the MTs. High speed centrifugation then spun down the MTs, leaving the kinesin in the supernatant. This kinesin was subjected to a centrifugal filtration using a 100 KD cut off filter for further purification, aliquoted, snap frozen in liquid nitrogen, and stored at -80 &deg;C. SDS gel electrophoresis and western blotting was performed using the purified sample. The motor activity of purified samples before and after the final centrifugal filtration step was evaluated using an in vitro single molecule microtubule assay. The kinesin fractions before and after the centrifugal filtration showed processivity as previously reported in literature. Further experiments are underway to evaluate the interaction between kinesin and other transport related proteins.

Isolation and Purification of Kinesin from Drosophila Embryos

This is a protocol to isolate active full length Kinesin from Drosophila embryos for single-molecule biophysical studies. We show how to collect embryos, make the embryo lysate, and then polymerize microtubules (MTs). Kinesin is purified by immobilizing it on the MTs, spinning down the Kinesin-MT complexes, and then releasing the kinesin from the MTs via ATP addition.

Isolamento e Purificação de cinesina partir Drosophila Os embriões

Motor proteins move cargos along microtubules, and transport them to specific sub-cellular locations. Because altered transport is suggested to underlie a ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

Vampiric Isolamento de fluido extracelular a partir de<em&gt; Caenorhabditis elegans</em

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an oil-coated graduated cylinder; landing height provided a measure of flight performance by assessing how far flies will fall before producing enough thrust to make contact with the wall of the cylinder. Here we describe an updated version of the flight tester with four major improvements. First, we added a &#34;drop tube&#34; to ensure that all flies enter the flight cylinder at a similar velocity between trials, eliminating variability between users. Second, we replaced the oil coating with removable plastic sheets coated in Tangle-Trap, an adhesive designed to capture live insects. Third, we use a longer cylinder to enable more accurate discrimination of flight ability. Fourth we use a digital camera and imaging software to automate the scoring of flight performance. These improvements allow for the rapid, quantitative assessment of flight behavior, useful for large datasets and large-scale genetic screens.

An Improved Method for Accurate and Rapid Measurement of Flight Performance in Drosophila

Here we describe a method for rapid and accurate measurement of flight performance in Drosophila, enabling high-throughput screening.

Um método melhorado para rápido e preciso de medição de desempenho em vôo<em&gt; Drosophila</em

Drosophila has proven to be a useful model system for analysis of behavior, including flight. The initial flight tester involved dropping flies into an ...

RNA Isolation from Mouse Pancreas: A Ribonuclease-rich Tissue

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

We report a procedure to isolate RNA with high integrity from the ribonuclease rich mouse pancreas.

Isolamento de RNA Rato Pâncreas: Um tecido rico em Ribonuclease

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge.  As a primary function of the pancreas is to aid ...

Hybrid-Cut: An Improved Sectioning Method for Recalcitrant Plant Tissue Samples

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some plant cells have rigid cell walls, tough fibers and crystals (calcium oxalate, silica, etc.), and high water content that often disrupt tissue integrity during plant tissue sectioning.
This study establishes a simple Hybrid-Cut tissue sectioning method. This protocol modifies a paraffin-based sectioning technique and improves the integrity of tissue sections from different plants. Plant tissues were embedded in paraffin before sectioning in a cryostat at -16 &#176;C. Sectioning under low temperature hardened the paraffin blocks, reduced tearing and scratching, and improved tissue integrity significantly. This protocol was successfully applied to calcium oxalate-rich Phalaenopsis orchid tissues as well as recalcitrant tissues such as reproductive organs and leaves of rice, maize, and wheat. In addition, the high quality of tissue sections from Hybrid-Cut could be used in combination with in situ hybridization (ISH) to provide spatial expression patterns of genes of interest. In conclusion, this protocol is particularly useful for recalcitrant plant tissue containing high crystal or silica content. Good quality tissue sections enable morphological and other biological studies.

This protocol describes a simple Hybrid-Cut tissue sectioning method that is useful for recalcitrant plant tissues. Good quality tissue sections enable anatomical studies and other biological studies including in situ hybridization (ISH).

Hybrid-Cut: Um Método Seccionamento melhorado para amostras de tecidos de plantas recalcitrante

Maintaining plant section integrity is essential for studying detailed anatomical structures at the cellular, tissue, or even organ level. However, some ...

Stomata Tape-Peel: An Improved Method for Guard Cell Sample Preparation

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is often difficult to isolate and thus studying them often provides a challenge. 
This study establishes a stomatal guard cell enrichment method. The protocol utilizes Scotch tape to isolate guard cells and extract proteins from the cells. This method improves the integrity and yield of guard cell during isolation and provides a reliable sample for the study of cell signaling processes and how they correlate to stomatal movement phenotypes. In this method, the plant leaves were partitioned between two portions of tape and peeled apart to remove the abaxial side. This protocol was applied to Arabidopsis leaf tissue to isolate guard cells. The cells were treated with fluorescein diacetate (FDA) to determine viability and with abscisic acid (ABA) to assess stomata movement in response to ABA. In conclusion, this protocol proves useful for preparing enriched stomatal guard cells and isolating proteins. The quality of the cells and proteins obtained here enable physiological and other biological studies.

This protocol describes a method of preparing enriched stomatal guard cells that is useful for physiological and other biological studies.

Fita de estomas-casca: Um método melhorado para preparação de amostras de células de guarda

The study of guard cells is essential to the knowledge of the specific contributions of this cell type to the overall function of plants. However, it is ...

An Improved Time- and Labor- Efficient Protocol for Mouse Primary Hepatocyte Isolation

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on different needs, like time, labor, cost, and primary hepatocyte usage, resulting in various primary hepatocyte isolation protocols. However, the numerous steps and time-consuming reagent preparations in primary hepatocyte isolation are major drawbacks for efficiency. After comparing different protocols for their pros and cons, the advantages of each were combined, and a rapid and efficient primary hepatocyte isolation protocol was formulated. Within only ~35 min, this protocol could yield as much, if not more, healthy primary hepatocytes as other protocols. Further, glucose metabolism experiments performed using the isolated primary hepatocytes validated the usefulness of this protocol in in vitro liver metabolism studies. We also extensively reviewed and analyzed the significance and purpose of each step in this study so that future researchers can further optimize this protocol based on needs.

Primary hepatocytes are a valuable tool to study liver response and metabolism in vitro. Utilizing commercially available reagents, an improved time- and labor-efficient protocol for mouse primary hepatocyte isolation was developed.

Um protocolo aprimorado de tempo e trabalho-eficiente para o isolamento primário do hepatocito do rato

Primary hepatocytes are used extensively in liver in vitro research, especially in glucose metabolism studies. A base technique has been adapted based on ...