Name: identification of circular rnas using rna sequencing
Uploaded: 2019-11-14T11:00:00
Description: Watch this Scientific Journal Video about Identification of Circular RNAs using RNA Sequencing at JoVE.com

Estamos agradecidos al Programa de Donación de Cerebros y Cuerpos del Instituto de Investigación de la Salud del Sol Banner (BBDP, por sus siglas en la) cuenta con el suministro de tejidos cerebrales humanos. El BBDP ha sido apoyado por el Instituto Nacional de Trastornos Neurológicos y Accidentes Cerebrovasculares (U24 NS072026 National Brain and Tissue Resource for Parkinson's Disease and Related Disorders), el National Institute on Aging (P30AG19610 Arizona Alzheimer's Disease Core Center), el Departamento de Servicios de Salud de Arizona (contrato 211002, Arizona Alzheimer's Research Center), la Comisión de Investigación Biomédica de Arizona (contratos 4001, 0011, 05-901 y 1001 al Consorcio de Enfermedad de Parkinson de Arizona) y el Consorcio de Enfermedadde Parkinson de Arizona) y el Consorcio Michael J. Fundación Fox para la Investigación de Parkinson27. Este estudio también fue apoyado por el DHS y el Estado de Arizona (aun vez ala DeDHS 14-052688). También agradecemos a Andrea Schmitt (Banner Research) y Cynthia Lechuga (TGen) por su apoyo administrativo.

Esta investigación se ha llevado a cabo de acuerdo con todas las directrices institucionales, nacionales e internacionales para el bienestar humano. Los tejidos cerebrales se obtuvieron del Programa de Donación cerebral y corporal del Instituto de Investigación de la Salud del Sol Banner en Sun City, AZ. Las operaciones del Programa de Donación cerebral y corporal son aprobadas por la Junta de Revisión Institucional de Occidente (protocolo #20120821). Todos los sujetos o sus representantes legales firmaron el consen...

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	<li>Salzman, J., Gawad, C., Wang, P. L., Lacayo, N., Brown, P. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circular+RNAs+are+the+predominant+transcript+isoform+from+hundreds+of+human+genes+in+diverse+cell+types.">Circular RNAs are the predominant transcript isoform from hundreds of human genes in diverse cell types.</a> PloS One. 7 (2), e30733 (2012).</li><li>Jeck, W. R., 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=Circular+RNAs+are+abundant,+conserved,+and+associated+with+ALU+repeats.">Circular RNAs are abundant, conserved, and associated with ALU repeats.</a> RNA. 19 (2), 141-157 (2013).</li><li>Memczak, S., 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=Circular+RNAs+are+a+large+class+of+animal+RNAs+with+regulatory+potency.">Circular RNAs are a large class of animal RNAs with regulatory potency.</a> Nature. 495 (7441), 333-338 (2013).</li><li>Sanger, H. L., Klotz, G., Riesner, D., Gross, H. J., Kleinschmidt, A. 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O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell-type+specific+features+of+circular+RNA+expression.">Cell-type specific features of circular RNA expression.</a> PLoS Genetics. 9 (9), e1003777 (2013).</li><li>Du, W. W., 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=Foxo3+circular+RNA+promotes+cardiac+senescence+by+modulating+multiple+factors+associated+with+stress+and+senescence+responses.">Foxo3 circular RNA promotes cardiac senescence by modulating multiple factors associated with stress and senescence responses.</a> European Heart Journal. 38 (18), 1402-1412 (2016).</li><li>Capel, B., 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=Circular+transcripts+of+the+testis-determining+gene+Sry+in+adult+mouse+testis.">Circular transcripts of the testis-determining gene Sry in adult mouse testis.</a> Cell. 73 (5), 1019-1030 (1993).</li><li>Hansen, T. B., 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=miRNA-dependent+gene+silencing+involving+Ago2-mediated+cleavage+of+a+circular+antisense+RNA.">miRNA-dependent gene silencing involving Ago2-mediated cleavage of a circular antisense RNA.</a> The EMBO Journal. 30 (21), 4414-4422 (2011).</li><li>Hansen, T. B., 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=Natural+RNA+circles+function+as+efficient+microRNA+sponges.">Natural RNA circles function as efficient microRNA sponges.</a> Nature. 495 (7441), 384-388 (2013).</li><li>Li, Z., 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=Exon-intron+circular+RNAs+regulate+transcription+in+the+nucleus.">Exon-intron circular RNAs regulate transcription in the nucleus.</a> Nature Structural &amp; Molecular Biology. 22 (3), 256-264 (2015).</li><li>Tan, W. L., 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=A+landscape+of+circular+RNA+expression+in+the+human+heart.">A landscape of circular RNA expression in the human heart.</a> Cardiovascular Research. 113 (3), 298-309 (2016).</li><li>Zhong, Z., Lv, M., Chen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Screening+differential+circular+RNA+expression+profiles+reveals+the+regulatory+role+of+circTCF25-miR-103a-3p/miR-107-CDK6+pathway+in+bladder+carcinoma.">Screening differential circular RNA expression profiles reveals the regulatory role of circTCF25-miR-103a-3p/miR-107-CDK6 pathway in bladder carcinoma.</a> Scientific Reports. 6, 30919 (2016).</li><li>Gao, Y., Wang, J., Zhao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CIRI:+an+efficient+and+unbiased+algorithm+for+de+novo+circular+RNA+identification.">CIRI: an efficient and unbiased algorithm for de novo circular RNA identification.</a> Genome Biology. 16, 4-014-0571-0573 (2015).</li><li>Wang, K., 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=MapSplice:+accurate+mapping+of+RNA-seq+reads+for+splice+junction+discovery.">MapSplice: accurate mapping of RNA-seq reads for splice junction discovery.</a> Nucleic Acids Research. 38 (18), e178 (2010).</li><li>Szabo, L., 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=Statistically+based+splicing+detection+reveals+neural+enrichment+and+tissue-specific+induction+of+circular+RNA+during+human+fetal+development.">Statistically based splicing detection reveals neural enrichment and tissue-specific induction of circular RNA during human fetal development.</a> Genome Biology. 16, 126-015-0690-0695 (2015).</li><li>Cheng, J., Metge, F., Dieterich, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Specific+identification+and+quantification+of+circular+RNAs+from+sequencing+data.">Specific identification and quantification of circular RNAs from sequencing data.</a> Bioinformatics. 32 (7), 1094-1096 (2016).</li><li>Zhang, X. O., 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=Complementary+sequence-mediated+exon+circularization.">Complementary sequence-mediated exon circularization.</a> Cell. 159 (1), 134-147 (2014).</li><li>Rybak-Wolf, 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=Circular+RNAs+in+the+mammalian+brain+are+highly+abundant,+conserved,+and+dynamically+expressed.">Circular RNAs in the mammalian brain are highly abundant, conserved, and dynamically expressed.</a> Molecular Cell. 58 (5), 870-885 (2015).</li><li>Ji, P., 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=Expanded+Expression+Landscape+and+Prioritization+of+Circular+RNAs+in+Mammals.">Expanded Expression Landscape and Prioritization of Circular RNAs in Mammals.</a> Cell Reports. 26 (12), 3444-3460 (2019).</li><li>Hansen, T. B., Ven&#248;, M. T., Damgaard, C. K., Kjems, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+circular+RNA+prediction+tools.">Comparison of circular RNA prediction tools.</a> Nucleic Acids Research. 44 (6), e58 (2016).</li><li>Zeng, X., Lin, W., Guo, M., Zou, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comprehensive+overview+and+evaluation+of+circular+RNA+detection+tools.">A comprehensive overview and evaluation of circular RNA detection tools.</a> PLoS Comput Biol. 13 (6), e1005420 (2017).</li><li>Sekar, S., 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=ACValidator:+a+novel+assembly-based+approach+for+in+silico+validation+of+circular+RNAs.">ACValidator: a novel assembly-based approach for in silico validation of circular RNAs.</a> bioRxiv. , (2019).</li><li>Westholm, J. O., 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=Genome-wide+analysis+of+drosophila+circular+RNAs+reveals+their+structural+and+sequence+properties+and+age-dependent+neural+accumulation.">Genome-wide analysis of drosophila circular RNAs reveals their structural and sequence properties and age-dependent neural accumulation.</a> Cell Reports. 9 (5), 1966-1980 (2014).</li><li>Szabo, L., Salzman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detecting+circular+RNAs:+bioinformatic+and+experimental+challenges.">Detecting circular RNAs: bioinformatic and experimental challenges.</a> Nature Reviews Genetics. 17 (11), 679-692 (2016).</li><li>Zheng, Y., Ji, P., Chen, S., Hou, L., Zhao, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reconstruction+of+full-length+circular+RNAs+enables+isoform-level+quantification.">Reconstruction of full-length circular RNAs enables isoform-level quantification.</a> Genome Medicine. 11 (1), 2 (2019).</li><li>Beach, T. G., 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=Arizona+study+of+aging+and+neurodegenerative+disorders+and+brain+and+body+donation+program.">Arizona study of aging and neurodegenerative disorders and brain and body donation program.</a> Neuropathology. 35 (4), 354-389 (2015).</li></ol>

En este estudio, se probaron dos kits de preparación de bibliotecas disponibles comercialmente, opciones de pretratamiento y cantidades de ARN de entrada con el fin de optimizar un protocolo de enriquecimiento de circRNA para la construcción de bibliotecas de secuenciación de circRNA. Sobre la base de las evaluaciones de este estudio, una serie de aspectos clave y pasos críticos en la creación de bibliotecas de secuenciación de circRNA son evidentes. Nuestra evaluación confirma la utilidad del pretratamiento RNase...

Los ARN circulares (ArnNA) son ARN endógenos y no codificantes que han ganado atención dada su expresión generalizada en el transcriptoma eucariota1,2,3. Se forman cuando los exons se empalman entre sí y por lo tanto se consideraron inicialmente como artefactos de empalme4,5. Sin embargo, estudios recientes han demostrado que los circRNAs exhiben tipo de célula, tejido y etapa específica del desarrollo expresión específica3,6 y se conservan evolutivamente2,3. Además, participan en la mediación de interacciones proteína-proteína7,la unión de microARN (miRNA)3,8,9,10, y la regulación de la transcripción genética parental11.
En la secuenciación clásica de ARN (RNA-seq), los círculos pueden perderse por completo durante la construcción de la biblioteca como resultado de la selección de poli-A para ARNm o pueden ser difíciles de aislar dada su baja abundancia. Sin embargo, estudios recientes de caracterización de circRNA han incorporado un paso de pretratamiento utilizando RNase R con el fin de enriquecer para circRNAs2,12,13. RNase R es una exoribonucleasa que digiere los ARN lineales, dejando atrás estructuras circulares de ARN. Los protocolos de enriquecimiento de CircRNA se optimizaron generando y comparando datos de dos kits de construcción de bibliotecas de transcriptomes completos disponibles comercialmente, con y sin un paso de pretratamiento RNase R, y utilizando cantidades variables de entrada total de ARN (1 a 4 g). El protocolo optimizado se utilizó a continuación para evaluar la abundancia de circRNAs en cinco regiones cerebrales diferentes (cerebelo [BC], lóbulo parietal inferior [IP], giso temporal medio [MG], corteza occipital [OC] y gros frontal superior [SF]) y otros cuatro tipos de tejido (hígado [LV], pulmón [LU], ganglio linfático [LN] y páncreas [PA]). Las bibliotecas RNA-seq se emparejaron secuenciadas finales y los datos se analizaron utilizando seis algoritmos de predicción de circRNA diferentes: find_circ3, CIRI14,Mapsplice15,KNIFE16, DCC17y CIRCexplorer18. Según nuestro análisis, se detectó el mayor número de circRNAs únicos cuando se utilizó un kit de preparación de la biblioteca de ARN total varado con pretratamiento RNase R y ARN de entrada total de 4 g. El protocolo optimizado se describe aquí. Como se informó anteriormente19,20, el mayor enriquecimiento de los círculos en el cerebro en comparación con otros tipos de tejido.

<table>
	<tbody>
		<tr>
			<td>1000 &#181;L pipette tips</td>
			<td>Rainin</td>
			<td>GP-L1000F</td>
			<td></td>
		</tr>
		<tr>
			<td>20 &#181;L pipette tips</td>
			<td>Rainin</td>
			<td>SR L 10F</td>
			<td></td>
		</tr>
		<tr>
			<td>200 &#181;L pipette tips</td>
			<td>Rainin</td>
			<td>SR L 200F</td>
			<td></td>
		</tr>
		<tr>
			<td>2200 TapeStation Accessories (foil covers)</td>
			<td>Agilent Technologies</td>
			<td>5067-5154</td>
			<td></td>
		</tr>
		<tr>
			<td>2200 TapeStation Accessories (tips)</td>
			<td>Agilent Technologies</td>
			<td>5067-5153</td>
			<td></td>
		</tr>
		<tr>
			<td>Adhesive Film for Microplates</td>
			<td>VWR</td>
			<td>60941-064</td>
			<td></td>
		</tr>
		<tr>
			<td>AMPure XP Beads 450 mL</td>
			<td>Beckman Coulter</td>
			<td>A63882</td>
			<td>PCR purification</td>
		</tr>
		<tr>
			<td>Eppendorf twin.tec 96-Well PCR Plates</td>
			<td>VWR</td>
			<td>951020401</td>
			<td></td>
		</tr>
		<tr>
			<td>High Sensitivity D1000 reagents</td>
			<td>Agilent Technologies</td>
			<td>5067-5585</td>
			<td></td>
		</tr>
		<tr>
			<td>High Sensitivity D1000 ScreenTape</td>
			<td>Agilent Technologies</td>
			<td>5067-5584</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 2500 Sequencing System</td>
			<td>Illumina</td>
			<td>SY-401-2501</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 3000/4000 PE Cluster Kit</td>
			<td>Illumina</td>
			<td>PE-410-1001</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 3000/4000 SBS Kit (150 cycles)</td>
			<td>Illumina</td>
			<td>FC-410-1002</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq 4000 Sequencing System</td>
			<td>Illumina</td>
			<td>SY-401-4001</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq PE PE Rapid Cluster Kit v2</td>
			<td>Illumina</td>
			<td>PE-402-4002</td>
			<td></td>
		</tr>
		<tr>
			<td>HiSeq Rapid SBS Kit v2 (50 cycle)</td>
			<td>Illumina</td>
			<td>FC-402-4022</td>
			<td></td>
		</tr>
		<tr>
			<td>Kapa Total RNA Kit</td>
			<td>Roche</td>
			<td>KK8400</td>
			<td></td>
		</tr>
		<tr>
			<td>Molecular biology grade ethanol</td>
			<td>Fisher Scientific</td>
			<td>BP28184</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit Assay Tubes</td>
			<td>Supply Center by Thermo Fischer</td>
			<td>Q32856</td>
			<td></td>
		</tr>
		<tr>
			<td>Qubit dsDNA High Sense Assay Kit</td>
			<td>Supply Center by Thermo Fischer</td>
			<td>Q32854</td>
			<td></td>
		</tr>
		<tr>
			<td>RNA cleanup and concentrator - 5</td>
			<td>Zymo</td>
			<td>RCC-100</td>
			<td>Contains purification columns, collection tubes</td>
		</tr>
		<tr>
			<td>RNAClean XP beads</td>
			<td>Beckman Coulter Genomics</td>
			<td></td>
			<td>RNA Cleanup beads</td>
		</tr>
		<tr>
			<td>Rnase R</td>
			<td>Lucigen</td>
			<td>RNR07250</td>
			<td></td>
		</tr>
		<tr>
			<td>SuperScript II Reverse Transcriptase 10,000 units</td>
			<td>ThermoFisher (LifeTech)</td>
			<td>18064014</td>
			<td></td>
		</tr>
		<tr>
			<td>TapeStation 2200</td>
			<td>Agilent Technologies</td>
			<td></td>
			<td>Nucleic Acid analyzer</td>
		</tr>
		<tr>
			<td>TElowE</td>
			<td>VWR</td>
			<td>10128-588</td>
			<td></td>
		</tr>
		<tr>
			<td>TruSeq Stranded Total RNA Library Prep Kit</td>
			<td>Illumina</td>
			<td>20020596</td>
			<td>Kit used in section 3</td>
		</tr>
		<tr>
			<td>Two-Compartment Divided Tray</td>
			<td>VWR</td>
			<td>3054-1004</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Water</td>
			<td>Supply Center by Thermo Fischer</td>
			<td>10977-015</td>
			<td></td>
		</tr>
		<tr>
			<td>Universal control RNA</td>
			<td>Agilent</td>
			<td>740000</td>
			<td></td>
		</tr>
	</tbody>
</table>

identification of circular rnas using rna sequencing

Los ARN circulares (circARN) son una clase de ARN no codificantes que participan en funciones que incluyen la regulación del microARN (miRNA), la mediación de interacciones proteínas y proteínas y la regulación de la transcripción genética parental. En la secuenciación clásica de ARN de próxima generación (RNA-seq), los círculos se pasan por alto típicamente como resultado de la selección de poli-A durante la construcción de bibliotecas de ARNm, o se encuentran en muy baja abundancia, y por lo tanto son difíciles de aislar y detectar. Aquí, se optimizó un protocolo de construcción de la biblioteca de circRNA comparando kits de preparación de bibliotecas, opciones de pretratamiento y varias cantidades totales de entrada de ARN. Se probaron dos kits de preparación de bibliotecas de transcriptomes completos disponibles en el uso comercial, con y sin pretratamiento de RNase R, y utilizando cantidades variables de entrada total de ARN (1 a 4 g). Por último, múltiples tipos de tejido; incluyendo hígado, pulmón, ganglio linfático y páncreas; así como múltiples regiones cerebrales; incluyendo el cerebelo, el lóbulo parietal inferior, el giso temporal medio, la corteza occipital y el giso frontal superior; se compararon para evaluar la abundancia de circARN a través de tipos de tejidos. El análisis de los datos de ARN-seq generados utilizando seis herramientas diferentes de detección de ARN (find_circ, CIRI, Mapsplice, KNIFE, DCC y CIRCexplorer) reveló que un kit de preparación de la biblioteca de ARN total varado con pretratamiento RNase R y entrada de ARN de 4 g es el óptimo método para identificar el mayor número relativo de circRNAs. De acuerdo con los hallazgos anteriores, se observó el mayor enriquecimiento de los circRNAs en los tejidos cerebrales en comparación con otros tipos de tejidos.

Los datos generados utilizando un ARN de control universal (UC) disponible comercialmente y utilizando dos kits de preparación de bibliotecas, que incluyen un paso de agotamiento de riboentos en sus protocolos, se evaluaron por primera vez. Utilizando un flujo de trabajo analítico (flujo de trabajo de análisis de datos, sección 4), en general, se detectó un mayor número de círculos en los conjuntos de datos TruSeq en comparación con los de Kapa (Figura 1). Aunque los porcentajes de A...

Watch this Scientific Journal Video about Identification of Circular RNAs using RNA Sequencing at JoVE.com

Identification of Circular RNAs using RNA Sequencing

Los ARN circulares (arnar) son ARN no codificantes que pueden tener funciones en la regulación transcripcional y en las interacciones mediantantes entre proteínas. Tras la evaluación de los diferentes parámetros para la construcción de bibliotecas de secuenciación de circRNA, se compiló un protocolo utilizando la preparación total de la biblioteca de ARN varada con el pretratamiento RNase R y se presenta aquí.

Identificación de ARN circulares mediante secuenciación de ARN

Circular RNAs (circRNAs) are a class of non-coding RNAs involved in functions including micro-RNA (miRNA) regulation, mediation of protein-protein ...

Este protocolo es el resultado de evaluar aspectos clave de la preparación de la biblioteca circRNA, como el tipo de kit, el tratamiento RNase R, las cantidades de entrada y su impacto en la detección de circRNA. Permite una detección óptima de circRNA y proporciona datos sobre parámetros ajustables en función de las necesidades del usuario. Identificar los circRNAs en diferentes tipos de muestras nos ayudará a comprender mejor la distribución de su expresión y establece el escenario para delinear el papel funcional de estas moléculas.Este método se puede aplicar a cualquier muestra total de ARN. Es importante mantener las muestras de ARN en hielo antes de comenzar el protocolo. Evalúe los valores RIN y DV200 en el Bioanalyzer Agilent o TapeStation antes de la preparación, y siga los procedimientos adecuados al trabajar con ARN.Comience diluyendo el ARN total a cuatro microgramos en 39 microlitros de agua libre de RNase y pipeteándolo arriba y abajo para mezclar. En un tubo separado, utilice 1 tampón RNase R para diluir la RNase R a una concentración de trabajo de dos unidades por microlitro. Pipeta 39 microlitros de ARN total y cinco microlitros de 10x RNase R Reaction Buffer en un tubo de reacción de 1,5 microlitros, y mezclar.Añadir seis microlitros de la RNase R.Then ajuste la pipeta al volumen de reacción completo, y mezcle la solución pipeteando hacia arriba y hacia abajo 10 veces. Coloque el tubo en un baño de agua Celsius de 37 grados durante 10 minutos, asegurándose de que todo el volumen de reacción esté sumergido en el baño de agua. A continuación, coloque el tubo sobre hielo e inmediatamente proceda con la limpieza y concentración de ARN.Antes de empezar, prepare el tampón de lavado de ARN mezclando el concentrado con 48 mililitros de 100% etanol, y coloque las columnas de purificación en los tubos de recolección. Cuando esté listo, agregue dos volúmenes de búfer de enlace de ARN a la muestra tratada con RNase R y mezcle bien. Agregue 150 microlitros de 100% etanol a la mezcla para un total de 300 microlitros, transfiera todo el volumen a la columna y centrifugar durante 30 segundos.Elimine el flujo a través y agregue 400 microlitros de RNA Prep Buffer directamente a la columna. Centrifugar durante 30 segundos y deseche el flujo a través. A continuación, agregue 700 microlitros de RNA Wash Buffer a la columna, centrifugar durante 30 segundos y deseche el flujo a través.Agregue otras 400 microlitros del wash Buffer, centrifugar durante dos minutos, y transfiera la columna a un tubo fresco de 1,5 mililitros sin RNase. Agregue cuidadosamente 11 microlitros de agua libre de RNase directamente a la columna sosteniendo la punta de la pipeta justo encima del filtro de columna. Deje reposar la muestra durante un minuto a temperatura ambiente y luego centrifugarlo durante un minuto.Asegúrese de que haya flujo a través del tubo de 1,5 mililitros y deseche la columna. La muestra de ARN purificado puede almacenarse a menos 80 grados centígrados o utilizarse inmediatamente para la preparación de la biblioteca. Transfiera 10 microlitros del ARN total purificado a un pozo limpio en una placa PCR de 96 pozos.Añadir cinco microlitros de rRNA Binding Buffer seguidos de cinco microlitros de rRNA Removal Mix, luego pipetear suavemente hacia arriba y hacia abajo para mezclar los reactivos. Selle la placa e ibloquela durante cinco minutos a 68 grados centígrados en un bloque termociclotro preprogramado y precalentado. Después de la incubación, deje reposar la placa a temperatura ambiente durante un minuto.Retire el sello de la placa y agregue 35 microlitros de perlas de extracción de ARN de temperatura ambiente con vórtices a la muestra. Ajuste la pipeta a 45 microlitros, y pipetee hacia arriba y hacia abajo de 10 a 20 veces para mezclar a fondo. Deje que la placa se siente a temperatura ambiente durante un minuto.Transfiera la placa a un soporte magnético e imagíe allí durante un minuto o hasta que la solución se despeje. Transfiera el sobrenadante a un nuevo pozo en el plato. A continuación, transfiera la placa del soporte magnético al soporte de la placa.Cuentas de limpieza de ARN vortex hasta que estén bien dispersas, y agregue 99 microlitros de cuentas a la muestra. Pipetear hacia arriba y hacia abajo para mezclar, e incubar la placa a temperatura ambiente durante 10 minutos. Transfiera la placa al soporte magnético, y déjela allí durante cinco minutos o hasta que la solución se aclare, luego retire y deseche todo el sobrenadante del pozo.Con la placa todavía en el soporte magnético, añadir 200 microlitros de 80% etanol recién preparado al pozo sin interrumpir las perlas. Deje el etanol en el pozo durante 30 segundos, luego retírelo y deséchelo. Agregue 11 microlitros de Elution Buffer al pozo, y pipetearlo arriba y abajo para mezclar.Incubar la placa a temperatura ambiente durante dos minutos, luego transferirla de vuelta al soporte magnético, y mantenerla allí hasta que la solución se despeje. Transfiera 8,5 microlitros del sobrenadante a un nuevo pozo en la misma placa, y agregue 8,5 microlitros de Elute, Primer, Fragment High Mix a cada pozo que contenga una muestra. Pipetear hacia arriba y hacia abajo 10 veces para mezclar bien, sellar la placa e incubar durante ocho minutos en un termociclador precalentado a 94 grados centígrados.Enfríe la muestra a cuatro grados centígrados, retire la placa del termociclo y centrifugar brevemente. Se evaluaron dos kits de preparación de bibliotecas, TruSeq y KAPA, para este protocolo. En general, se detectó un mayor número de ARN circulares y un menor porcentaje de ARN ribosomal cuando se utiliza el kit TruSeq, por lo que TruSeq fue seleccionado para otros experimentos.La importancia del pretratamiento de RNase R se evaluó comparando los datos generados a partir de bibliotecas pretratadas y no pretratadas. Como era de esperar, se identificó sistemáticamente un mayor número de ARN circulares en las bibliotecas pretratadas en comparación con las no pretratadas. La cantidad de ARN de entrada se optimizó para detectar una mayor diversidad de ARN circulares.Las bibliotecas se prepararon utilizando uno, dos y cuatro microgramos de ARN de entrada, y se observó la mayor diversidad de especies de ARN circular cuando se utilizaban cuatro microgramos de ARN total. El protocolo optimizado se utilizó para comparar las abundancias circulares de ARN en cinco regiones cerebrales de cuatro donantes sanos y para otros tipos de tejidos de seis donantes sanos. En general, se observó una mayor abundancia de ARNa circular en el cerebro en comparación con otros tipos de tejido.Es importante recordar seguir los procedimientos apropiados cuando se trabaja con ARN, incluyendo el uso de guantes, limpiar a fondo el banco y las pipetas con toallitas RNase o aerosol antes de comenzar el protocolo, y mantener el ARN en hielo de antemano. Después de este procedimiento, se puede realizar la validación ortogonal como la secuenciación de Sanger de cruces de circRNA identificados. El descubrimiento de nuevos circRNAs y más evidencia de su presencia tallan una nueva área de investigación para explorar sus funciones biológicas.

identification-of-circular-rnas-using-rna-sequencing

Research

JoVE Journal

Genetics

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Mapeo de las interacciones RNA-ARN a nivel mundial usando Psoralen Biotinilado

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Investigación

Genética

Metabolic Labeling and Profiling of Transfer RNAs Using Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for protein synthesis by translating codons in mRNA into amino acid sequences. tRNAs were long considered as house-keeping molecules that lacked regulatory functions. However, a growing body of evidence indicates that cellular tRNA levels fluctuate in correspondence to varying conditions such as cell type, environment, and stress. The fluctuation of tRNA expression directly influences gene translation, favoring or repressing the expression of particular proteins. Ultimately comprehending the dynamic of protein synthesis requires the development of methods able to deliver high-quality tRNA profiles. The method that we present here is named SPOt, which stands for Streamlined Platform for Observing tRNA. SPOt consists of three steps starting with metabolic labeling of cell cultures with radioactive orthophosphate, followed by guanidinium thiocyanate-phenol-chloroform extraction of radioactive total RNAs and finally hybridization on in-house printed macroarrays. tRNA levels are estimated by quantifying the radioactivity intensities at each probe spot. In the protocol presented here we profile tRNAs in Mycobacterium smegmatis mc2155, a nonpathogenic bacterium often used as a model organism to study tuberculosis.

We describe an original transfer RNA analysis platform named SPOt (Streamlined Platform for Observing tRNA). SPOt simultaneously measures cellular levels of all tRNAs in biological samples, in only three steps, and in less than 24 hours.

Etiquetado metabólico y perfiles de RNAs de transferencia utilizando Macroarrays

Transfer RNAs (tRNA) are abundant short non-coding RNA species that are typically 76 to 90 nucleotides in length. tRNAs are directly responsible for ...

Retrospective MicroRNA Sequencing: Complementary DNA Library Preparation Protocol Using Formalin-fixed Paraffin-embedded RNA Specimens

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of cancer development. By using non-invasive or pre-malignant lesions from patients who later develop invasive disease, gene expression analyses may help identify early molecular alterations that predispose to cancer risk. It has been well described that nucleic acids recovered from FFPE tissues have undergone severe physical damage and chemical modifications, which make their analysis difficult and generally requires adapted assays. MicroRNAs (miRNAs), however, which represent a small class of RNA molecules spanning only up to ~18&#8211;24 nucleotides, have been shown to withstand long-term storage and have been successfully analyzed in FFPE samples. Here we present a 3&#39; barcoded complementary DNA (cDNA) library preparation protocol specifically optimized for the analysis of small RNAs extracted from archived tissues, which was recently demonstrated to be robust and highly reproducible when using archived clinical specimens stored for up to 35 years. This library preparation is well adapted to the multiplex analysis of compromised/degraded material where RNA samples (up to 18) are ligated with individual 3&#39; barcoded adapters and then pooled together for subsequent enzymatic and biochemical preparations prior to analysis. All purifications are performed by polyacrylamide gel electrophoresis (PAGE), which allows size-specific selections and enrichments of barcoded small RNA species. This cDNA library preparation is well adapted to minute RNA inputs, as a pilot polymerase chain reaction (PCR) allows determination of a specific amplification cycle to produce optimal amounts of material for next-generation sequencing (NGS). This approach was optimized for the use of degraded FFPE RNA from specimens archived for up to 35 years and provides highly reproducible NGS data.

Formalin-fixed paraffin-embedded specimens represent a valuable source of molecular biomarkers of human diseases. Here we present a laboratory-based cDNA library preparation protocol, initially designed with fresh frozen RNA, and optimized for the analysis of archived microRNAs from tissues stored up to 35 years.

La secuencia retrospectiva MicroRNA: ADN complementario biblioteca preparación protocolo utilizando a muestras de ARN de parafina-encajado formalina-fijo

&#8211;Archived, clinically classified formalin-fixed paraffin-embedded (FFPE) tissues can provide nucleic acids for retrospective molecular studies of ...

Rare Event Detection Using Error-corrected DNA and RNA Sequencing

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been used to analyze the spectrum of clonal mutations in malignancy. Though far more efficient than traditional Sanger methods, NGS struggles with identifying rare clonal and subclonal mutations due to its high error rate of ~0.5&#8211;2.0%. Thus, standard NGS has a limit of detection for mutations that are &#62;0.02 variant allele fraction (VAF). While the clinical significance for mutations this rare in patients without known disease remains unclear, patients treated for leukemia have significantly improved outcomes when residual disease is &#60;0.0001 by flow cytometry. In order to mitigate this artefactual background of NGS, numerous methods have been developed. Here we describe a method for Error-corrected DNA and RNA Sequencing (ECS), which involves tagging individual molecules with both a 16 bp random index for error-correction and an 8 bp patient-specific index for multiplexing. Our method can detect and track clonal mutations at variant allele fractions (VAFs) two orders of magnitude lower than the detection limit of NGS and as rare as 0.0001 VAF.

Next-generation sequencing (NGS) is a powerful tool for genomic characterization that is limited by the high error rate of the platform (~0.5&#8211;2.0%). We describe our methods of error-corrected sequencing that allow us to obviate the NGS error rate and detect mutations at variant allele fractions as rare as 0.0001.

Detección de eventos raros usando ADN Error corregido y secuenciación de ARN

Conventional next-generation sequencing techniques (NGS) have allowed for immense genomic characterization for over a decade. Specifically, NGS has been ...

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

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

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

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

Captura de alto rendimiento identificación de secuencias reguladoras de genes utilizando secuenciación de próxima generación de conformación Circular cromosoma (4C-seq)

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

Overexpressing Long Noncoding RNAs Using Gene-activating CRISPR

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since 2007. These studies have confirmed that lncRNAs are altered in almost all diseases. However, studying the functional roles for lncRNAs in the context of disease remains difficult due to the lack of protein products, tissue-specific expression, low expression levels, complexities in splice forms, and lack of conservation among species. Given the species-specific expression, lncRNA studies are often restricted to human research contexts when studying disease processes. Since lncRNAs function at the molecular level, one way to dissect lncRNA biology is to either remove the lncRNA or overexpress the lncRNA and measure cellular effects. In this article, a written and visualized protocol to overexpress lncRNAs in vitro is presented. As a representative experiment, an lncRNA associated with inflammatory bowel disease, Interferon Gamma Antisense 1 (IFNG-AS1), is shown to be overexpressed in a Jurkat T-cell model. To accomplish this, the activating clustered regularly interspaced short palindromic repeats (CRISPR) technique is used to enable overexpression at the endogenous genomic loci. The activating CRISPR technique targets a set of transcription factors to the transcriptional start site of a gene, enabling a robust overexpression of multiple lncRNA splice forms. This procedure will be broken down into three steps, namely (i) guide RNA (gRNA) design and vector construction, (ii) virus generation and transduction, and (iii) colony screening for overexpression. For this representative experiment, a greater than 20-fold enhancement in IFNG-AS1 in Jurkat T cells was observed.

Traditional cDNA-based overexpression techniques have a limited applicability for the overexpression of long noncoding RNAs due to their multiple splice forms with potential functionality. This review reports a protocol using CRISPR technology to overexpress multiple splice variants of a long noncoding RNA.

Overexpressing larga Noncoding RNAs mediante activación de gen CRISPR

Long noncoding RNA (lncRNA) biology is a new and exciting field of research, with the number of publications from this field growing exponentially since ...

Sequencing of mRNA from Whole Blood using Nanopore Sequencing

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, and was deployed to West Africa during the recent Ebola virus epidemic, highlighting this possibility. In addition to its practical advantages (low cost, ease of equipment transport and use), this technology also provides fundamental advantages over second-generation sequencing approaches, particularly the very long read length, ability to directly sequence RNA, and real-time availability of data. Raw read accuracy is lower than with other sequencing platforms, which represents the main limitation of this technology; however, this can be partially mitigated by the high read depth generated. Here, we present a field-compatible protocol for sequencing of the mRNAs encoding for Niemann-Pick C1, which is the cellular receptor for ebolaviruses. This protocol encompasses extraction of RNA from animal blood samples, followed by RT-PCR for target enrichment, barcoding, library preparation, and the sequencing run itself, and can be easily adapted for use with other DNA or RNA targets.

Nanopore sequencing is a novel technology that allows cost-effective sequencing in remote locations and resource-poor settings. Here, we present a protocol for sequencing of mRNAs from whole blood that is compatible with such conditions.

Secuenciación de ARNm de sangre entera utilizando secuenciación de nanoporos

Sequencing in remote locations and resource-poor settings presents unique challenges. Nanopore sequencing can be successfully used under such conditions, ...

Single-cell RNA Sequencing and Analysis of Human Pancreatic Islets

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to normal and pathological conditions. The goal of this protocol is to generate high-quality, large-scale transcriptome data of each endocrine cell type with the use of a droplet-based microfluidic single-cell RNA sequencing technology. Such data can be utilized to build the gene expression profile of each endocrine cell type in normal or specific conditions. The process requires careful handling, accurate measurement, and rigorous quality control. In this protocol, we describe detailed steps for human pancreatic islets dissociation, sequencing, and data analysis. The representative results of about 20,000 human single islet cells demonstrate the successful application of the protocol.

Here, we present a protocol to generate high-quality, large-scale transcriptome data of single cells from isolated human pancreatic islets using a droplet-based microfluidic single-cell RNA sequencing technology.

Secuenciación y análisis de islotes pancreáticos humanos de una sola célula

Pancreatic islets comprise of endocrine cells with distinctive hormone expression patterns. The endocrine cells show functional differences in response to ...

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT&#39;s advantages and applications and discuss some of its limitations.

Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing RIPiT-Seq

Here, we present a protocol to enrich endogenous RNA binding sites or &#34;footprints&#34;&#160;of RNA:protein (RNP) complexes from mammalian cells. This approach involves two immunoprecipitations of RNP subunits and is therefore dubbed RNA immunoprecipitation in tandem (RIPiT).

Identificación de Huellas de ARN:Complejos proteicos a través de la inmunoprecipitación de ARN en tándem seguido de secuenciación (RIPiT-Seq)

RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT ...

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as plant microRNAs (miRNAs) carry a 2&#39;-O-methyl (2&#39;-OMe) modification at their 3&#39;&#160;terminal nucleotide. This modification adds another difficulty as it inhibits 3&#39;&#160;adapter ligation. We previously demonstrated that modified versions of the &#39;TruSeq (TS)&#39;&#160;protocol have less bias and an improved detection of 2&#39;-OMe RNAs. Here we describe in detail protocol &#39;TS5&#39;, which showed the best overall performance. TS5 can be followed either using homemade reagents or reagents from the TS kit, with equal performance.

Improving Small RNA-seq: Less Bias and Better Detection of 2&#039;-O-Methyl RNAs

We present a detailed small RNA library reparation protocol with less bias than standard methods and an increased sensitivity for 2&#39;-O-methyl RNAs. This protocol can be followed using homemade reagents to save cost or using kits for convenience.

Mejora de ARN pequeño-seq: Menos sesgo y mejor detección de 2'-O-Metil RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...