This work was supported by the Swiss National Science Foundation Sinergia grant CRSII3 147697, the Stiftung f&#252;r Angewandte Krebsforschung and Z&#252;rich Krebsliga.

1. Preparation of Cytosolic and Nuclear Protein Fraction
<ol>
	<li>Plate 4 x 106 ACC-MESO-4 cells in a T150 cell culture flask grown in RPMI - 1640 medium supplemented with 10% FBS, 1x penicillin/streptomycin (100x), and 2 mM L-Glutamine (200 mM). When cells reach a confluence of 80-90% (~5-5.5 x 106 cells), proceed with protein extraction of nuclear and cytosolic fraction. 
	NOTE: ACC-MESO-4 cell line was obtained from the RIKEN BioResource Centre11.</li>
	<li>Aspirat...

<ol>
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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. 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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. 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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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The authors declare that they have no competing financial interests.

3&#39; UTRs belong to the non-coding genome3, and all non-coding RNAs can interact with proteins in order to exert their function7. When the mammalian genome was found to be pervasively transcribed and produced a significant portion of long noncoding RNAs16, emerging evidences demonstrated that these long-noncoding RNAs function in regulating gene expression as they interact with chromatin-remodeling complexes17. This knowledg...

Gene expression and the final level of the gene product can be tightly regulated by affecting the mRNA stability and mRNA translation rate1. These post-transcriptional regulatory mechanisms are exerted through the interactions of non-coding RNA and/or RNA-binding proteins (RBPs) with targeted mRNA. It is usually the 3&#39; untranslated region of mRNA (3&#39; UTR - belonging to the non-coding portion of the genome2) that contains specific cis-regulatory elements (CRE), which are recognized by trans-acting factors such as miRNA or RBPs3. The best-studied cis-element within the 3&#39; UTR, is the adenine-rich element (ARE) motif, which is recognized by specific AU-binding proteins (AUBP), and, in turn, induces either mRNA degradation/deadenylation (ARE-mediated decay) or mRNA stabilization4.
The size of the 3&#39; UTR of calretinin mRNA (CALB2) is 573 bp long and contains a putative AUUUA pentamer, as predicted by AREsite2, a bioinformatic tool5. Consistent with the presence of a putative ARE motif, the pmirGLO vector-reporter assay demonstrated a stabilization role of this element within CALB2 mRNA6. Finally, the in vitro RNA-pulldown was used to identify the AUBP that stabilizes calretinin mRNA through the ARE motif.
Since all non-coding RNAs interact with proteins7, the in vitro RNA-pulldown is a good way and first-of-choice assay for identifying RNA-interactors to aid in deciphering molecular mechanisms. In this RNA-pulldown method, commercially synthesized RNA probes, which were labeled with a modified form of biotin (desthiobiotin) at the 3&#39; terminus of the RNA strand, were used. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the bound-RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. Such RNA-bound proteins are eluted from the magnetic beads, run through a 12% SDS-PAGE gel, transferred to a membrane, and probed with different antibodies.
In the standard streptavidin-biotin affinity purification procedure, harsh denaturation conditions are required to disrupt the strong irreversible biotin-streptavidin bond to elute the bound proteins8, which could lead to the dissociation of protein complexes. Unlike biotin, desthiobiotin reversibly binds to streptavidin and is competitively displaced with a buffered solution of biotin, allowing for the gentle elution of proteins, and avoiding the isolation of naturally biotinylated molecules9, suggesting that the technique is ideal for isolating native protein complexes under native conditions.
In vitro binding conditions and stringency, which are determined by salt concentration, reducing agents and detergent percentage, should be close to those present in the cellular context in order to identify true in vivo interactions. The binding conditions implemented herein have been previously demonstrated as appropriate for the identification of the HuR as an AU-binding protein10. This approach could save time, since optimization of proper binding conditions can be time-consuming and challenging. In addition, this method could be used as a starting protocol for any RNA-pulldown experiment and can be gradually optimized by changing the concentration of salts and detergents, changing the glycerol percentage, and adding other salts. Moreover, we demonstrated that even a short 25-nucleotide RNA-probe harboring a pentamer ARE-motif could be used to demonstrate interaction with a specific AUBP.

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

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 structures and motifs. In this RNA-pulldown protocol, commercially synthesized RNA probes are labeled with a modified form of biotin, desthiobiotin, at the 3' terminus of the RNA strand, which reversibly binds to streptavidin and thus allows elution of proteins under more physiological conditions. The RNA-desthiobiotin is immobilized through interaction with streptavidin on magnetic beads, which are used to pull down proteins that specifically interact with the RNA of interest. Non-denatured and active proteins from the cytosolic fraction of mesothelioma cells are used as the source of proteins. The method described here can be applied to detect the interaction between known RNA binding proteins and a 25-nucleotide (nt) long RNA probe containing a sequence of interest. This is useful to complete the functional characterization of stabilizing or destabilizing elements present in RNA molecules achieved using a reporter vector assay.

In this experiment, a 25-nt long fragment of calretinin 3&#39; UTR harboring ARE motif (CALB2 3&#39; UTR (ARE) 25-nt) was used to test whether it binds specifically to the Human-antigen R (HuR) protein, a known mRNA stabilizer. To test the specificity of the ARE element, a 25-nt RNA probe CALB2 3&#39; UTR (mtARE) containing an ARE-motif mutation, which was previously shown to abolish the stabilization effect of the ARE motif, was used6. The third RNA probe represen...

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

Desthiobiotin labeling of a synthetic 25-nucleotide RNA oligo, which contains an adenine-rich element (ARE) motif, allows specific binding of cytosolic ARE-binding protein.

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.

desthiobiotin-streptavidin-affinity-mediated-purification-rna

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7.8K vues.  University Hospital Zurich. 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 t...