Name: desthiobiotin-streptavidin-affinity mediated purification of rna-interacting proteins in mesothelioma cells
Uploaded: 2018-04-25T15:00:00
Description: Watch this Scientific Journal Video about Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells at JoVE.com

Este trabajo fue apoyado por la concesión de la Sinergia de la Fundación de ciencia nacional suizo CRSII3 147697, la Stiftung für Angewandte Krebsforschung y Zürich Krebsliga.

1. preparación de la fracción de proteínas citosólicas y nucleares
<ol><li>Placa 4 x 106 ACC-MESO-4 células en un frasco de cultivo celular T150 cultivadas en RPMI - 1640 medio suplementado con 10% FBS, 1 x penicilina/estreptomicina (100 x) y 2 mM L-glutamina (200 mM). Cuando las células alcanzan una confluencia del 80-90% (~5-5.5 x 106 células), proceder a la extracción de proteínas de la fracción nuclear y citosólica. Nota: Línea de celular de ACC-MESO-4 se obtuvo de la RIKEN BioR...

<ol>
	<li>Glisovic, T., Bachorik, J. L., Yong, J., Dreyfuss, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNA-binding+proteins+and+post-transcriptional+gene+regulation.">RNA-binding proteins and post-transcriptional gene regulation.</a> FEBS Letters. 582 (14), 1977-1986 (2008).</li><li>Mayr, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Evolution+and+biological+roles+of+alternative+3'UTRs.">Evolution and biological roles of alternative 3'UTRs.</a> Trends in Cell Biology. 26 (3), 227-237 (2016).</li><li>Matoulkova, E., Michalova, E., Vojtesek, B., Hrstka, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+3'+untranslated+region+in+post-transcriptional+regulation+of+protein+expression+in+mammalian+cells.">The role of the 3' untranslated region in post-transcriptional regulation of protein expression in mammalian cells.</a> RNA Biology. 9 (5), 563-576 (2012).</li><li>von Roretz, C., Di Marco, S., Mazroui, R., Gallouzi, I. 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L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=AREsite2:+An+enhanced+database+for+the+comprehensive+investigation+of+AU/GU/U-rich+elements.">AREsite2: An enhanced database for the comprehensive investigation of AU/GU/U-rich elements.</a> Nucleic Acids Research. 44, D90-D95 (2016).</li><li>Kresoja-Rakic, 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=Posttranscriptional+regulation+controls+calretinin+expression+in+malignant+pleural+mesothelioma.">Posttranscriptional regulation controls calretinin expression in malignant pleural mesothelioma.</a> Frontiers in Genetics. 8 (70), (2017).</li><li>Chu, C., Spitale, R. C., Chang, H. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Technologies+to+probe+functions+and+mechanisms+of+long+noncoding+RNAs.">Technologies to probe functions and mechanisms of long noncoding RNAs.</a> Nature Structural &amp; Molecular Biology. 22 (1), 29-35 (2015).</li><li>Diamandis, E. P., Christopoulos, T. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+biotin-(strept)avidin+system:+principles+and+applications+in+biotechnology.">The biotin-(strept)avidin system: principles and applications in biotechnology.</a> Clinical Chemistry. 37 (5), 625-636 (1991).</li><li>Hirsch, J. D., 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=Easily+reversible+desthiobiotin+binding+to+streptavidin,+avidin,+and+other+biotin-binding+proteins:+Uses+for+protein+labeling,+detection,+and+isolation.">Easily reversible desthiobiotin binding to streptavidin, avidin, and other biotin-binding proteins: Uses for protein labeling, detection, and isolation.</a> Analytical Biochemistry. 308 (2), 343-357 (2002).</li><li>. Magnetic bead technology for better assay development Available from: <a target="_blank" href="https://assets.thermofisher.com/TFS-Assets/LSG/brochures/1602577-Magnetic-Bead-Technology-Brochure.pdf">https://assets.thermofisher.com/TFS-Assets/LSG/brochures/1602577-Magnetic-Bead-Technology-Brochure.pdf</a> (2013)</li><li>Usami, N., 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=Establishment+and+characterization+of+four+malignant+pleural+mesothelioma+cell+lines+from+Japanese+patients.">Establishment and characterization of four malignant pleural mesothelioma cell lines from Japanese patients.</a> Cancer Science. 97 (5), 387-394 (2006).</li><li>Laemmli, U. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cleavage+of+structural+proteins+during+the+assembly+of+the+head+of+bacteriophage+T4.">Cleavage of structural proteins during the assembly of the head of bacteriophage T4.</a> Nature. 227 (5259), 680-685 (1970).</li><li>Coulson, R. M., Cleveland, D. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ferritin+synthesis+is+controlled+by+iron-dependent+translational+derepression+and+by+changes+in+synthesis/transport+of+nuclear+ferritin+RNAs.">Ferritin synthesis is controlled by iron-dependent translational derepression and by changes in synthesis/transport of nuclear ferritin RNAs.</a> Proceedings of the National Academy of Sciences of the United States of America. 90 (16), 7613-7617 (1993).</li><li>Leibold, E. A., Munro, H. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoplasmic+protein+binds+in+vitro+to+a+highly+conserved+sequence+in+the+5'+untranslated+region+of+ferritin+heavy-+and+light-subunit+mRNAs.">Cytoplasmic protein binds in vitro to a highly conserved sequence in the 5' untranslated region of ferritin heavy- and light-subunit mRNAs.</a> Proceedings of the National Academy of Sciences of the United States of America. 85 (7), 2171-2175 (1988).</li><li>Srikantan, S., Gorospe, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HuR+function+in+disease.">HuR function in disease.</a> Frontiers in Bioscience (Landmark Edition). 17, 189-205 (2012).</li><li>Morris, K. V., Mattick, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+rise+of+regulatory+RNA.">The rise of regulatory RNA.</a> Nature Reviews: Genetics. 15 (6), 423-437 (2014).</li><li>Rinn, J. 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=Functional+demarcation+of+active+and+silent+chromatin+domains+in+human+HOX+loci+by+noncoding+RNAs.">Functional demarcation of active and silent chromatin domains in human HOX loci by noncoding RNAs.</a> Cell. 129 (7), 1311-1323 (2007).</li><li>Zhao, J., Sun, B. K., Erwin, J. A., Song, J. J., Lee, J. 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3' UTRs pertenecen al genoma no codificante3, y todos los RNAs no codificantes pueden interactuar con proteínas para ejercer su función7. Cuando el genoma mamífero fue encontrado para ser transcrito lo impregna y produjeron una porción significativa de larga noncoding RNAs16, nuevas evidencias demostraron que estos larga noncoding RNAs función en la regulación de expresión génica al interactuar con remodelación de cromatina complejos<sup cla...

Expresión génica y el nivel final del producto del gene pueden ser reguladas fuertemente afectando la estabilidad del ARNm y ARNm traducción tarifa1. Estos mecanismos de regulación postranscripcional se ejercen a través de las interacciones de no codificante del RNA y RNA-que ata las proteínas (restrictivas) con el mRNA específicos. Generalmente es el 3' región sin traducir de mRNA (3' UTR - pertenecientes a la parte no codificante del genoma2) que contiene específico cis-elementos reguladores (CRE), que son reconocidos por trans-actúan factores como miRNA o restrictivas 3. el mejor estudio cis-elemento dentro de los 3' UTR, es el adorno de elementos ricos en adenina (son), que es reconocido por proteínas específicas AU (AUBP) y, a su vez, induce o mRNA degradación/deadenylation (mediada son decaimiento) o mRNA estabilización4.
El tamaño de los 3' UTR del calretinin mRNA (CALB2) es 573 bp largo y contiene una supuesta AUUUA pentámero, como AREsite2, una herramienta bioinformática del5. Consistente con la presencia de un supuesto motivo son, el análisis de vector-reportero de pmirGLO había demostrado un papel de estabilización de este elemento dentro de mRNA CALB26. Por último, el en vitro RNA-telecine se utilizó para identificar la AUBP que estabiliza calretinin mRNA mediante el adorno son.
Pues todo no-codificación RNAs interactúan con proteínas7, el en vitro RNA-telecine es una buena forma y análisis de primera de elección para la identificación de RNA-interactianos a ayudar a descifrar los mecanismos moleculares. En este método de RNA-telecine, comercialmente sintetizadas sondas de RNA, que fueron etiquetadas con una forma modificada de la biotina (desthiobiotin) en la terminal 3' de la hebra de RNA, fueron utilizadas. El RNA-desthiobiotin se inmoviliza mediante la interacción con estreptavidina en granos magnéticos, que se utilizan para tirar abajo proteínas que interaccionan específicamente con el limite-RNA de interés. Proteínas no desnaturalizadas y activas de la fracción citosólica de las células del mesotelioma se utilizan como fuente de proteínas. Tales proteínas RNA-bound se eluyen de las bolas magnéticas, a través de un gel de SDS-PAGE de 12%, transferidos a una membrana y sondeado con los diferentes anticuerpos.
En el procedimiento de purificación de afinidad estándar de estreptavidina-biotina, desnaturalización dura condiciones se requieren interrumpir el vínculo fuerte biotina-estreptavidina irreversible para eluir las proteínas límite8, que podría conducir a la disociación de complejos de la proteína. A diferencia de la biotina, desthiobiotin se une a estreptavidina reversible y se desplaza competitivamente con una solución de biotina, lo que permite la elución suave de proteínas y evitar el aislamiento de naturalmente biotinilado moléculas9 lo que sugiere que la técnica es ideal para el aislamiento de complejos de proteína nativa en condiciones nativas.
In vitro condiciones obligatorias y rigor, que están determinadas por la concentración de sales, agentes reductores y porcentaje de detergente, deben estar cerca de los presentes en el contexto celular con el fin de identificar interacciones de verdad en vivo . Las condiciones vinculantes en este documento han sido previamente demostradas según corresponda para la identificación de lo HuR como una AU-que atan la proteína10. Este enfoque podría ahorrar tiempo, ya que la optimización de las condiciones de enlace adecuada puede ser difícil y desperdiciador de tiempo. Además, este método podría utilizarse como un protocolo inicial para cualquier experimento de RNA-desplegable y puede optimizarse gradualmente cambiando la concentración de sales y detergentes, cambiando el porcentaje de glicerol y adición de otras sales. Por otra parte, hemos demostrado que incluso una corto 25-nucleótido RNA sonda albergando un pentámero son motivo podía utilizarse para demostrar la interacción con una AUBP específico.

<table border="0" cellpadding="0" cellspacing="0" width="1340">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">NE-PER Nuclear and cytoplasmic extraction kit</td>
			<td style="width:295px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:124px;">78833</td>
			<td style="width:468px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce Protease Inhibitor Tablets, EDTA-free</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:124px;">A32965</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pierce BCA Protein Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pierce Magnetic RNA-protein Pull-Down Kit&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">20164</td>
			<td>Includes magnetic beads and therefore the kit need to be stored at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Pierce RNA 3&#8217; End Desthiobiotinylation Kit&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">20163</td>
			<td style="width:468px;">The kit is a part of the &#34;Pierce Magnetic RNA-protein Pull-Down Kit&#34;, and needs to be stored at -20 &#176;C unlike the pull-down kit (4 &#176;C).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-2,&#160; magnetic stand</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">12321D</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:453px;">Anti - HuR (MOUSE) monoclonal antibody</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">-</td>
			<td style="width:468px;">The antibody is included in the Pierce Magnetic RNA-protein Pull-Down Kit&#160; - 1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - &#945; - tubulin (MOUSE) monoclonal antibody</td>
			<td>Santa Cruz</td>
			<td style="width:124px;">8035</td>
			<td>1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - PARP (RABBIT) Polyclonal antibody</td>
			<td>Cell Signaling</td>
			<td>9542</td>
			<td>1:1000 in 5% BSA 1x TTBS - goat anti-rabbit secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - Mesothelin (MOUSE) Monoclonal Antibody&#160;</td>
			<td>Rockland</td>
			<td>200-301-A87</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Secondary goat anti-rabbit antibody</td>
			<td style="width:295px;">Cell Signaling</td>
			<td style="width:124px;">7074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Secondary rabbit anti-mouse antibody</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A-9044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RPMI - 1640 medium</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">FBS - Filtrated Bovine Serum</td>
			<td style="width:295px;">Pan Biotech&#160;</td>
			<td style="width:124px;">P40-37500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Penicillin - streptomycin (100x)</td>
			<td style="width:295px;">Sigma-Aldrich&#160;</td>
			<td style="width:124px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">L-Glutamine solution (200 mM)</td>
			<td style="width:295px;">Sigma-Aldrich&#160;</td>
			<td style="width:124px;">G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">0.25% Trypsin-EDTA (1x)</td>
			<td style="width:295px;">Gibco by Life technologies&#160;</td>
			<td style="width:124px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN 3 Cell</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653301</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini Protean System Glass Plates&#160;</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN Spacer Plates with 1.5 mm integrated spacer</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN Comb, 10-well, 1.5 mm, 66 &#181;L</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-PROTEAN Tetra Cell Casting Modules</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1658050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNaseZap RNase Decontamination Solution</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:453px;">Rotiphorese gel 30 - aqueous 30% acrylamide and bisacrylamide stock solution at a ration of 37.5:1</td>
			<td style="width:295px;">Roth</td>
			<td style="width:124px;">3029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">20% SDS&#160;</td>
			<td style="width:295px;">PanReac AppliChem</td>
			<td>A3942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">PVDF transfer membrane&#160;</td>
			<td style="width:295px;">Perkin Elmer</td>
			<td style="width:124px;">NEF1002</td>
			<td>Need to be activated by incubating in pure methanol for 1 min followed by washing in water for 2 min&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Trans-Blot SD semi-dry transfer cell&#160;</td>
			<td style="width:295px;">Bio-Rad</td>
			<td>1703940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Clarity Western ECL Substrate</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1705061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">FusionFX&#160; Digital Imager</td>
			<td style="width:295px;">Vilber</td>
			<td style="width:124px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNA oligo synthesis</td>
			<td style="width:295px;">Mycrosynth AG, Switzerland</td>
			<td style="width:124px;">-</td>
			<td>the synthesis scale - 0.04 &#181;mol - HPLC purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Bovine serum albumin</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Hydrochloric Acid (HCl) 6 M</td>
			<td style="width:295px;">PanReac AppliChem</td>
			<td style="width:124px;">182883.1211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Ultra Pure 1 M Tris, pH 7.5&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">15567027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Glycerol&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td>17904</td>
			<td>50% sterile aliquots to be stored at -20&#176;C</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:453px;">Pierce Streptavidin magnetic beads&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">88817</td>
			<td style="width:468px;">Please note that these magnetic beads have an average diameter of 1 &#181;m (range from 0.5 - 1.5 &#181;m). Furthermore, Dynabeads-M280 Streptavidin, which are beads of homogenouse size 2.8 &#181;m, are also used in RNA-pulldown but be aware that this may affect purification process.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">TEMED</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Ammonium persulfate&#160;</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Coomassie G-250</td>
			<td style="width:295px;">Applichem</td>
			<td style="width:124px;">A3480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminiumsulfate-(14-18)-hydrate&#160;</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">368458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ortho-phosphoric acid&#160;</td>
			<td style="width:295px;">Applichem</td>
			<td style="width:124px;">A0637</td>
			<td></td>
		</tr>
	</tbody>
</table>

desthiobiotin-streptavidin-affinity mediated purification of rna-interacting proteins in mesothelioma cells

El en vitro RNA-telecine se utiliza todavía en gran parte en los primeros pasos de protocolos dirigidos a identificar proteínas de unión de ARN que reconocen determinados motivos y estructuras de ARN. En este protocolo de RNA-telecine, comercialmente sintetizadas sondas de RNA están marcadas con una forma modificada de la biotina, desthiobiotin, en el 3' terminal del filamento del RNA, que se une reversiblemente a estreptavidina y permite así la elución de proteínas bajo más fisiológica condiciones. El RNA-desthiobiotin se inmoviliza mediante la interacción con estreptavidina en granos magnéticos, que se utilizan para tirar abajo proteínas que interaccionan específicamente con el RNA de interés. Proteínas no desnaturalizadas y activas de la fracción citosólica de las células del mesotelioma se utilizan como fuente de proteínas. El método aquí descrito puede aplicarse para detectar la interacción entre proteínas RNA conocidas y una sonda de RNA largo (nt) de 25 nucleótidos que contiene una secuencia de interés. Esto es útil para completar la caracterización funcional de estabilizar o desestabilizar elementos presentes en las moléculas de ARN alcanzadas usando un análisis de vectores de reportero.

En este experimento, un fragmento largo 25-nt de calretinin 3' UTR que es motivo (CALB2 3' UTR (son) 25-nt) fue utilizado para probar si se une específicamente a la proteína del antígeno humano R (HuR), un conocido estabilizador de mRNA. Para probar la especificidad del elemento son, un RNA 25 nt sonda CALB2 3' UTR (mtARE) que contiene una mutación son Adorno, que previamente fue demostrada para suprimir el efecto de estabilización del motivo son, fue usado6. ...

Watch this Scientific Journal Video about Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells at JoVE.com

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Desthiobiotin-estreptavidina-afinidad mediada por purificación de proteínas de interacción con RNA en células de mesotelioma

Desthiobiotin etiquetado de un sintético 25-nucleótido RNA oligo, que contiene un motivo de elementos ricos en adenina (ARE), permite la unión específica de proteína citosólica son.

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

The overall goal of this procedure is to capture cytosolic protein that bind to a specific sequence located in RNA molecule in mesothelioma cells. This method can help answer key question in RNA biology field, such as if a predicted RNA binding protein can bind adenosine or uridine-rich sequence in a transcript. The main advantage of this technique is that it complements functional characterization of stabilizing or destabilizing elements assessed by reporter assay.Demonstrating this technique will be Dr.Jelena Kresoja-Rakic, a postdoc from my laboratory. To begin the protocol, obtain previously prepared ACC-MESO-4 cells with a 80 to 90%confluence. Aspirate the medium, wash the cells with 15 milliliters of 1X phosphate buffered saline, or PBS, and aspirate the PBS.Next, add three milliliters of 0.25%trypsin-EDTA, and incubate the culture flask at 37 degrees Celsius for three minutes. Add seven milliliters of supplemented RPMI-1640 medium, collect the cells in a 15-milliliter tube, and spin them down at 200 times g for five minutes. Then, aspirate the medium, and resuspend the cell pellet with 1.5 milliliters of 1X PBS.Then, transfer the cells into a 1.5-milliliter microcentrifuge tube. Spin down the cells again at 500 times g and four degrees Celsius for five minutes. After the spin, discard the supernatant, resuspend the cell pellet with 1.5 milliliters of ice-cold 1X PBS, and spin down the cells at 500 times g and four degrees Celsius for three minutes.Discard the supernatant, and estimate the cell pellet packed volume by comparing the tube containing the cell pellet with a 1.5-milliliter tube with 10, 20, 50, and 100 microliters of 1X PBS. Add 200 microliters of ice-cold cytoplasmic extraction reagent, or CER I, supplemented with two microliters of 1X proteinase inhibitor. Vigorously vortex the pellet for 15 seconds, and incubate the tube on ice for 10 minutes.Add 11 microliters of ice-cold cytoplasmic extraction reagent II, or CER II, vigorously vortex for five seconds, and incubate the tube for one minute on ice. Vortex the sample vigorously for five seconds, and centrifuge the tube at 16, 000 times g for five minutes. Transfer the supernatant to the clean, pre-chilled tube on ice.Resuspend the remaining pellet in 100 microliters of ice-cold nuclear extraction reagent, or NER, supplemented with one microliter of proteinase inhibitor, and vigorously vortex for 15 seconds. Incubate the sample on ice for 40 minutes, and vortex every 10 minutes for 15 seconds. Centrifuge the tube at 16, 000 times g for 10 minutes.Transfer the supernatant, which is the nuclear protein fraction, to a clean, pre-chilled tube. Immediately measure the protein concentration using the bicinchoninic method. Then, prepare 25 microliters of aliquots of cytosolic and nuclear extracts.Snap-freeze these aliquots by placing them in liquid nitrogen for five seconds, and store them directly at minus 80 degrees Celsius. Transfer five microliters of 10-micromolar RNA probes into 0.5-milliliter, thin-wall microcentrifuge tubes, and incubate them in a PCR machine at 85 degrees Celsius for five minutes. Place the tubes immediately on ice.For a single 30-microliter reaction, add 10 microliters of previously prepared mix to the RNA-containing tube. Carefully add 15 microliters of PEG 30%to the reaction, and use a fresh tip to mix the reaction. Incubate the reaction overnight at 16 degrees Celsius.The next day, prepare the reagents. Add 70 microliters of nuclease-free water to the RNA-labeling reaction tubes. Add 100 microliters of chloroform-isoamyl alcohol, and vortex briefly and spin the sample at 13, 000 times g for three minutes.Carefully remove only the upper phase. Avoid touching the lower phase. Transfer it to a new nuclease-free, 1.5-milliliter tube.Add 10 microliters of five-molar sodium chloride, one microliter of glycogen, and 300 microliters of ice-cold 100%ethanol. Place the tube at minus 20 degrees Celsius for two hours. After two hours, centrifuge at 13, 000 times g and four degrees Celsius for 15 minutes.Carefully discard the supernatant without disturbing the pellet. Add 300 microliters of ice-cold 70%ethanol, and centrifuge again at 13, 000 times g and four degrees Celsius for five minutes. Discard the supernatant completely, and air-dry the pellet for 15 minutes.Resuspend the pellet in 20 microliters of nuclease-free water. Incubate the RNA at 90 degrees Celsius for two minutes, and place it on ice. Place the labeled RNA on ice during the following step of pre-washing of the streptavidin magnetic beads.Prewash 50 microliters of streptavidin magnetic beads per 50 picomoles of RNA. Vortex the tube with streptavidin magnetic beads for 15 seconds. After vortexing quickly, remove 200 microliters into a clean, 1.5-milliliter Safe-Lock tube using cut pipette tips.Next, place the tube on a magnetic stand so that the beads collect at the side of the tube, and wait one minute. Remove the resuspension liquid. To wash the beads, remove the tube from the magnetic stand, add 400 microliters of 0.1-molar sodium hydroxide, 0.05-molar sodium chloride solution, and gently pipette up and down several times.Place the tube back on the magnetic stand, and wait one minute. Then, collect the supernatant. Wash the beads with 200 microliters of 100-millimolar sodium chloride, and remove the supernatant.Add 200 microliters of 20-millimolar tris, resuspend the beads by pipetting, and place the tube on the magnetic stand. After one minute, remove the supernatant. Then, remove the tube from the magnetic stand, add 200 microliters of 1X RNA capture buffer, and resuspend streptavidin magnetic beads by briefly vortexing.Remove 50 microliters of streptavidin magnetic beads, and add it to each labeled RNA tube using a cut pipette tip. Incubate the tubes for 30 minutes at room temperature on a roller. After the tube has been on the magnetic stand for one minute, remove the supernatant.Add 50 microliters of 20-millimolar tris to the beads, and resuspend them. Then, place the tubes into the magnetic stand. After one minute, remove the supernatant.Add 100 microliters of 1X protein RNA binding buffer to the beads, and resuspend them. Then, place the tubes back into the magnetic stand. Once the tubes have been in the stand for one minute, collect the supernatant.Add 100 microliter of master mix B, resuspend by gently pipetting up and down, and avoid creating bubbles. Incubate reaction tubes for 60 minutes at four degrees Celsius on a roller. Move on to the wash and elution steps.Place the tubes into a magnetic stand, collect the flow through, and transfer it into a new tube. Pipette 100 microliters of 1X wash buffer on the beads, resuspend gently, place them back into the stand, wait one minute, and discard the supernatant. Repeat this step two times.Add 40 microliters of elution buffer to the magnetic beads, mix by pipetting up and down, and incubate on a tube shaker. Place the tubes into a magnetic stand, wait one minute, and collect eluate sample. Place on ice, and use for downstream analysis.To demonstrate the purity of the nuclear/cytosolic fractions, proteins were analyzed by Western blot, which showed that alpha-tubulin was only detected in the cytosolic fraction, and PARP protein was detected only in the nuclear fraction. The eluate from the CALB2 three-prime UTR ARE probe demonstrated binding of human R and was absent in the eluate from the mutant probe and the unrelated RNA probe, which binds the iron-responsive protein. To further demonstrate the specificity between the CALB three-prime UTR and human R, the membrane was probed with anti-mesothelin antibody, and all three eluate samples showed no presence of mesothelin, indicating that the stabilizing ARE motif within CALB2 three-prime UTR can specifically bind human R protein.Coomassie staining of the gel shows that equal amounts of proteins were used to incubate with the three different RNA probes. Following this procedure, additional method protein-centric, like immunoprecipitation of a given protein, can be performed in order to address additional question, like which sequence bind to it. After its development, this technique paved the way for researchers in the field of RNA biology to explore possible interacting partners of a specific RNA.

Research

JoVE Journal

Cancer Research

Counting Proteins in Single Cells with Addressable Droplet Microarrays

Recuento de proteínas en las células con Microarrays de gota direccionable

Often cellular behavior and cellular responses are analyzed at the population level where the responses of many cells are pooled together as an average ...

Investigación

Investigación sobre el cáncer

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Un modelo de ratón preclínica de Osteosarcoma a definir la comunicación mediada por vesículas extracelular entre Tumor y las células madre mesenquimales

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Functional Assessment of BRCA1 variants using CRISPR-Mediated Base Editors

Evaluación funcional de variantes BRCA1 utilizando editores base mediados por CRISPR

Recent studies have investigated the risks associated with BRCA1 gene mutations using various functional assessment methods such as fluorescent reporter ...

In Vitro Evaluation of Oncogenic Transformation in Human Mammary Epithelial Cells

Evaluación In Vitro de la Transformación Oncogénica en Células Epiteliales Mamarias Humanas

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Fluorescence Microscopy for ATP Internalization Mediated by Macropinocytosis in Human Tumor Cells and Tumor-xenografted Mice

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Generation and Expansion of Primary, Malignant Pleural Mesothelioma Tumor Lines

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Current methodologies for the expansion of primary tumor cell lines from rare tumor types are lacking. This protocol describes methods to expand primary ...

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Ex Vivo Organoid Model of Adenovirus-Cre Mediated Gene Deletions in Mouse Urothelial Cells

Ex Vivo Modelo organoide de deleciones genéticas mediadas por adenovirus-Cre en células uroteliales de ratón

Bladder cancer is an understudied area, particularly in genetically engineered mouse models (GEMMs). Inbred GEMMs with tissue-specific Cre and loxP sites ...

Simultaneous Imaging and Flow-Cytometry-based Detection of Multiple Fluorescent Senescence Markers in Therapy-Induced Senescent Cancer Cells

Detección simultánea basada en imágenes y citometría de flujo de múltiples marcadores de senescencia fluorescente en células cancerosas senescentes inducidas por terapia

Chemotherapeutic drugs can induce irreparable DNA damage in cancer cells, leading to apoptosis or premature senescence. Unlike apoptotic cell death, ...

Modeling Brain Metastasis by Internal Carotid Artery Injection of Cancer Cells

Modelado de metástasis cerebrales mediante inyección de células cancerosas en la arteria carótida interna

Brain metastasis is a cause of severe morbidity and mortality in cancer patients. Critical aspects of metastatic diseases, such as the complex neural ...

Orthotopic Implantation of Patient-Derived Cancer Cells in Mice Recapitulates Advanced Colorectal Cancer

La implantación ortotópica de células cancerosas derivadas de pacientes en ratones recapitula el cáncer colorrectal avanzado

Over the last decade, more sophisticated preclinical colorectal cancer (CRC) models have been established using patient-derived cancer cells and 3D ...