Name: an efficient method for the isolation of highly purified rna from seeds for use in quantitative transcriptome analysis
Uploaded: 2017-01-11T13:00:00
Description: Watch this Scientific Journal Video about An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis at JoVE.com

Agradecemos à equipe de Genômica Funcional Facility e Espectrografia e BioImaging Facility, Nibb Núcleo Instalações de pesquisa, e para o modelo de pesquisa de plantas, Nibb Bioresource Center.

1. Extracção do ARN total de sementes de plantas <ol><li> Prepare conjuntos de buffer, colunas de spin, 1,5 e 2,0 tubos de polipropileno ml, e tubos de 1,5 mL de polipropileno livre de nuclease. </li><li> Adicionar 1% (w / v) de polivinilpirrolidona de grau de biologia molecular (daqui em diante referida como PVP) para a célula de tampão de lise para extracção de ARN e vortex vigorosamente. Incubar durante 20 min a 25 ° C para dissolver completamente. Após 20 min de incubação, misturam suavemente o tampão, ...

<ol>
	<li>Hills, M. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+storage-product+synthesis+in+seeds.">Control of storage-product synthesis in seeds.</a> Curr Opin Plant Biol. 7 (3), 302-308 (2004).</li><li>Li-Beisson, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Acyl-lipid+metabolism.">Acyl-lipid metabolism.</a> Arabidopsis Book. 11, e0161 (2013).</li><li>Bates, P. D., Stymne, S., Ohlrogge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biochemical+pathways+in+seed+oil+synthesis.">Biochemical pathways in seed oil synthesis.</a> Curr Opin Plant Biol. 16 (3), 358-364 (2013).</li><li>Santos-Mendoza, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deciphering+gene+regulatory+networks+that+control+seed+development+and+maturation+in+Arabidopsis.">Deciphering gene regulatory networks that control seed development and maturation in Arabidopsis.</a> Plant J. 54 (4), 608-620 (2008).</li><li>Durrett, T. P., Benning, C., Ohlrogge, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plant+triacylglycerols+as+feedstocks+for+the+production+of+biofuels.">Plant triacylglycerols as feedstocks for the production of biofuels.</a> Plant J. 54 (4), 593-607 (2008).</li><li>Kanai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Plastidic+DEAD-box+RNA+helicase+22,+HS3,+is+essential+for+plastid+functions+both+in+seed+development+and+in+seedling+growth.">The Plastidic DEAD-box RNA helicase 22, HS3, is essential for plastid functions both in seed development and in seedling growth.</a> Plant Cell Physiol. 54 (9), 1431-1440 (2013).</li><li>Kanai, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extension+of+oil+biosynthesis+during+the+mid-phase+of+seed+development+enhances+oil+content+in+Arabidopsis+seeds.">Extension of oil biosynthesis during the mid-phase of seed development enhances oil content in Arabidopsis seeds.</a> Plant Biotechnol J. 14 (5), 1241-1250 (2016).</li><li>Dekkers, B. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+reference+genes+for+RT-qPCR+expression+analysis+in+Arabidopsis+and+tomato+seeds.">Identification of reference genes for RT-qPCR expression analysis in Arabidopsis and tomato seeds.</a> Plant Cell Physiol. 53 (1), 28-37 (2012).</li><li>Salzman, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+improved+RNA+isolation+method+for+plant+tissues+containing+high+levels+of+phenolic+compounds+or+carbohydrates.">An improved RNA isolation method for plant tissues containing high levels of phenolic compounds or carbohydrates.</a> Plant Mol Biol Rep. 17 (1), 11-17 (1999).</li><li>Vicient, C. M., Delseny, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+total+RNA+from+Arabidopsis+thaliana+seeds.">Isolation of total RNA from Arabidopsis thaliana seeds.</a> Anal Biochem. 268 (2), 412-413 (1999).</li><li>Wang, G. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+high+quality+RNA+from+cereal+seeds+containing+high+levels+of+starch.">Isolation of high quality RNA from cereal seeds containing high levels of starch.</a> Phytochem Analysis. 23 (2), 159-163 (2012).</li><li>Birtic, S., Kranner, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+high-quality+RNA+from+polyphenol-,+polysaccharide-+and+lipid-rich+seeds.">Isolation of high-quality RNA from polyphenol-, polysaccharide- and lipid-rich seeds.</a> Phytochem Analysis. 17 (3), 144-148 (2006).</li></ol>

perfis de expressão gênica contribuir para a nossa compreensão da fisiologia da planta; Assim, os métodos de isolamento de ARN específico foram desenvolvidas de cada amostra condição 9-12. Foram investigados os processos que foram inibidas durante o isolamento de ARN a partir de sementes e descobriram que o RNA se ligar a membranas de sílica foi severamente inibido. Grandes quantidades de óleo, proteínas e polifenóis inibem isolamento do RNA. Nós modificámos o processo de extracção de ARN para ...

As plantas produzem sementes, que dão origem à próxima geração. Sementes acumular grandes quantidades de reservas de armazenagem, tais como óleos, hidratos de carbono e proteínas, para o crescimento pós-germinativo. Os seres humanos utilizar as reservas de armazenamento de sementes como fontes de alimentos e ração animal, e, portanto, sementes de plantas são um dos principais fornecedores de matéria orgânica comestível em todo o mundo. Aumentar a produção de sementes é um desafio importante na ciência das plantas. Desde reservas de conservação das sementes são fontes de valor comercial de alimentos e materiais industriais, os mecanismos moleculares subjacentes à regulação do metabolismo destas reservas têm sido amplamente investigadas 1-6. Além disso elucidação desses mecanismos será útil para aumentar a produção de sementes de culturas. Sementes se desenvolvem nos ovários de plantas após a fertilização, e eles amadurecem através de uma série de estágios de desenvolvimento 1,6,7. Para compreender melhor o desenvolvimento mecanismo molecular subjacente de sementes requer detalhada, Perfis de expressão de gene precisas de uma série de desenvolvimento de sementes a ser produzido. No entanto, as elevadas quantidades de óleos, proteínas, hidratos de carbono, e polifenóis em sementes de plantas tornar-se difícil de isolar ARN altamente purificada, o que se opõe perfil preciso da expressão do gene. Aqui, nós apresentamos um método eficiente para o isolamento de ARN a partir de sementes oleaginosas que contenham grandes quantidades de óleos, proteínas e polifenóis. Usando este método, os pesquisadores serão capazes de preparar RNA altamente purificada. Tal RNA será útil para monitorar mudanças de transcrição dos genes-chave que controlam a regulação metabólica das reservas de armazenagem de sementes no desenvolvimento e oleaginosas maduras.

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	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">RNeasy Plant Mini Kit</td>
			<td style="width:81px;">QIAGEN</td>
			<td style="width:136px;">74904</td>
			<td style="width:191px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">polyvinylpyrrolidone</td>
			<td style="width:81px;">Sigma-Aldrich</td>
			<td style="width:136px;">P5288-100G</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:247px;">HOMOGENIZER S-303</td>
			<td style="width:81px;">AS ONE</td>
			<td style="width:136px;">1-1133-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">NanoDrop Lite</td>
			<td style="width:81px;">Thermo Scientific</td>
			<td style="width:136px;">ND-NDL-US-CAN</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">PrimeScript RT reagent Kit (Perfect Real Time)</td>
			<td style="width:81px;">TAKARA</td>
			<td style="width:136px;">RR037A</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:247px;">KAPA SYBR Fast qPCR kit</td>
			<td style="width:81px;">Kapa biosystems</td>
			<td style="width:136px;">KK4601</td>
			<td></td>
		</tr>
	</tbody>
</table>

an efficient method for the isolation of highly purified rna from seeds for use in quantitative transcriptome analysis

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as industrial materials. Gene expression profiles are powerful tools for investigating metabolic regulation in plant cells. Therefore, detailed, accurate gene expression profiles during seed development are required for crop breeding. Acquiring highly purified RNA is essential for producing these profiles. Efficient methods are needed to isolate highly purified RNA from seeds. Here, we describe a method for isolating RNA from seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method enables highly purified RNA to be obtained from seeds without the use of phenol, chloroform, or additional processes for RNA purification. This method is applicable to Arabidopsis, rapeseed, and soybean seeds. Our method will be useful for monitoring the expression patterns of low level transcripts in developing and mature seeds.

Em primeiro lugar, investigou a melhor concentração de PVP usando Arabidopsis sementes maduras. O ARN total foi isolado a partir de cerca de 1000 sementes de acordo com o protocolo descrito acima, utilizando tampão de lise celular contendo 0%, 0,25%, 0,5%, 1,0% ou 2,0% de PVP. Após homogeneização e centrifugação, o sobrenadante foi recolhido, evitando a camada de óleo e detritos de sementes (Figura 1A). <p class="jove_content" fo:keep-together.withi...

Watch this Scientific Journal Video about An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis at JoVE.com

An Efficient Method for the Isolation of Highly Purified RNA from Seeds for Use in Quantitative Transcriptome Analysis

We have succeeded in establishing a method for RNA isolation from plant seeds containing large amounts of oils, proteins, and polyphenols, which have inhibitory effects on high-purity RNA isolation. Our method is suitable for monitoring the expression of genes with low level transcripts in seeds.

Um método eficiente para o isolamento de ARN altamente purificada a partir de sementes para Utilização em Análise Quantitativa do transcriptoma

Plant seeds accumulate large amounts of storage reserves comprising biodegradable organic matter. Humans rely on seed storage reserves for food and as ...

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