Name: adapting 3' rapid amplification of cdna ends to map transcripts in cancer
Uploaded: 2018-03-28T15:00:00
Description: Watch this Scientific Journal Video about Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer at JoVE.com

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<ol><li>Wachsen die HeLa-Zelllinie und zwei Aussetzung Mantel Zelle Lymphom Ze...

<ol>
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W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+amplification+of+3&#8242;+cDNA+ends+(3&#8242;-RACE).">Rapid amplification of 3&#8242; cDNA ends (3&#8242;-RACE).</a> Cold Spring Harb Protoc. 2006 (1), (2006).</li><li>Masamha, C. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+amplification+of+cDNA+ends.">Rapid amplification of cDNA ends.</a> PCR Cloning Protocols: Methods in Molecular Biology. 67, 233-238 (1997).</li><li>Freeman, L. A., Freeman, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cloning+full-length+transcripts+and+transcript+variants+using+5'+and+3'+RACE.">Cloning full-length transcripts and transcript variants using 5' and 3' RACE.</a> Lipoproteins and Cardiovascular Disease: Methods in Molecular Biology (Methods and Protocols). 1027, 3-17 (2013).</li><li>Masamha, C. P., Albrecht, T. R., Wagner, E. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+characterization+of+a+novel+CCND1/MRCK+gene+fusion+in+mantle+cell+lymphoma.">Discovery and characterization of a novel CCND1/MRCK gene fusion in mantle cell lymphoma.</a> J Hematol Oncol. 9 (1), 1-5 (2016).</li><li>Parker, B. C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+tumorigenic+FGFR3-TACC3+gene+fusion+escapes+miR-99a+regulation+in+glioblastoma.">The tumorigenic FGFR3-TACC3 gene fusion escapes miR-99a regulation in glioblastoma.</a> J Clin Investig. 123 (2), 855-865 (2013).</li><li>Prakash, T., 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=Expression+of+conjoined+genes:+Another+mechanism+for+gene+regulation+in+eukaryotes.">Expression of conjoined genes: Another mechanism for gene regulation in eukaryotes.</a> PLOS ONE. 5 (10), e13284 (2010).</li><li>Mertens, F., Johansson, B., Fioretos, T., Mitelman, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+emerging+complexity+of+gene+fusions+in+cancer.">The emerging complexity of gene fusions in cancer.</a> Nat Rev Cancer. 15 (6), 371-381 (2015).</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 Cell Biol. 26 (3), 227-237 (2016).</li><li>International Committee For Standardization In, H., 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=Protocol+for+evaluation+of+automated+blood+cell+counters.">Protocol for evaluation of automated blood cell counters.</a> Clin Lab Haematol. 6 (1), 69-84 (1984).</li><li>Desjardins, P., Conklin, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NanoDrop+microvolume+quantitation+of+nucleic+acids.">NanoDrop microvolume quantitation of nucleic acids.</a> J Vis Exp. (45), e2565 (2010).</li><li>Lorenz, T. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Polymerase+chain+reaction:+Basic+protocol+plus+troubleshooting+and+optimization+strategies.">Polymerase chain reaction: Basic protocol plus troubleshooting and optimization strategies.</a> J Vis Exp. (63), e3998 (2012).</li><li>Lee, P. Y., Costumbrado, J., Hsu, C. -. Y., Kim, Y. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agarose+gel+electrophoresis+for+the+separation+of+DNA+fragments.">Agarose gel electrophoresis for the separation of DNA fragments.</a> J Vis Exp. (62), e3923 (2012).</li><li>Curtis, P. C., Thomas, J. L., Phillips, N. R., Roby, R. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+primer+specific+filter+metrics+for+the+assessment+of+mitochondrial+DNA+sequence+data.">Optimization of primer specific filter metrics for the assessment of mitochondrial DNA sequence data.</a> Mitochondrial DNA. 21 (6), 191-197 (2010).</li><li>Letowski, J., Brousseau, R., Masson, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Designing+better+probes:+Effect+of+probe+size,+mismatch+position+and+number+on+hybridization+in+DNA+oligonucleotide+microarrays.">Designing better probes: Effect of probe size, mismatch position and number on hybridization in DNA oligonucleotide microarrays.</a> J Microbiol Methods. 57 (2), 269-278 (2004).</li><li>Lefever, S., Pattyn, F., Hellemans, J., Vandesompele, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-nucleotide+polymorphisms+and+other+mismatches+reduce+performance+of+quantitative+PCR+assays.">Single-nucleotide polymorphisms and other mismatches reduce performance of quantitative PCR assays.</a> Clin Chem. 59 (10), 1470-1480 (2013).</li><li>Singh, V. K., Govindarajan, R., Naik, S., Kumar, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+hairpin+structure+on+PCR+amplification+efficiency.">The effect of hairpin structure on PCR amplification efficiency.</a> Mol Biol Today. 1, 67-69 (2000).</li><li>Kalle, E., Kubista, M., Rensing, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multi-template+polymerase+chain+reaction.">Multi-template polymerase chain reaction.</a> Biomol Detect Quant. 2, 11-29 (2014).</li><li>Stransky, N., Cerami, E., Schalm, S., Kim, J. L., Lengauer, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+landscape+of+kinase+fusions+in+cancer.">The landscape of kinase fusions in cancer.</a> Nat Commun. 5, 4846 (2014).</li></ol>

Trotz des Aufkommens der massiven parallelen Sequenziertechnologien auf Basis gen von gen bleibt 3' Rennen immer noch die einfachste und wirtschaftlichste Methode zur Identifizierung der PAS und Nukleotide angrenzend an das Poly-Heck. Die hier beschriebene Anpassung erweitert mit 3' Rennen sowohl verstärken als auch Karte Sequenzen, die einen Teil des ORF, das Stopp-Codon und die gesamte 3' UTR der ANKHD1 mRNA Abschrift enthalten. Ein großer Vorteil von 3' RACE ist, dass Produkte von 3' Rennen mit ein paar kle...

Die Bildung von 3'-Ende ist ein entscheidender Schritt in mRNA-Reifung, die besteht aus der Spaltung der Pre-mRNA flussabwärts ein Pas, gefolgt von dem Zusatz von ~ 250 Untemplated Adenines, die Poly Tail1,2ausmachen. Poly-Bindeprotein (PABP) bindet an das Poly-Heck, und das schützt die mRNA-Transkript vor Abbau und erleichtert die Übersetzung1.
Aktuelle Schätzungen gehen davon aus, dass 70 % der menschlichen Gene mehrere PASs haben und somit alternative Polyadenylation, wodurch mehrere 3' Enden3zu unterziehen. So ist es wichtig zu erkennen, wo die Poly-Rute für den Rest der 3' UTR legt, sowie identifizieren die PAS von jeder gegebenen Protokoll verwendet. Das Aufkommen der Sequenzierung der nächsten Generation führte die gleichzeitige Identifikation von 3' wo und den PASs von tausend Genen. Dieser Anstieg der Sequenzierung Fähigkeit erforderte die Entwicklung von der bioinformatische Algorithmen für die Datenanalyse mit alternative Polyadenylation vom 3'-Ende. Für die de-Novo -Erkennung oder Validierung des PAS und damit des 3' Endes einzelner Gene von großflächigen Sequenzierungsdaten mapping bleibt 3' Rennen die Methode der Wahl4,5. Die Sequenzen in cDNA-Produkte von 3' Rennen enthalten normalerweise enthalten nur einen Teil der 3' UTR, die Poly-Tail, die Spaltstelle, PAS und die Sequenzen stromaufwärts des PAS enthält. Im Gegensatz zu PCR, wonach die Gestaltung und Nutzung von Gen spezifische forward und reverse Primer benötigt 3' Rennen nur zwei Gen spezifische verschachtelte forward Primer. PCR erfordert daher eine genauere Kenntnis der Nukleotidsequenz einer großen Region des Gens wird verstärkt4,6. Da 3' Rennen verwendet umgekehrt das gleiche Grundierung, dass Ziele der poly(A) tail für alle Polyadenylated RNA-Transkripte, nur die forward Primer gen spezifische, müssen somit nur erfordern Kenntnisse über eine deutlich kleinere Region der mRNA. Dies ermöglicht die Verstärkung der Regionen, deren Sequenzen nicht vollständig charakterisiert4,7. Dadurch konnten 3' Rennen verwendet werden, nicht nur um festzustellen, das 3'-Ende eines Gens, sondern um auch zu bestimmen und zu charakterisieren, große Regionen oberhalb des PAS, die einen erheblichen Teil der 3' UTR bilden. Durch die Kombination von 5' Rennen mit der modifizierten 3' Rennen, die größere Teile der 3' UTR und flankierenden Regionen umfasst, ist es möglich, vollständig sequenzieren oder Klonen eine gesamte mRNA-Transkript von 5'-Ende, deren Ende 3'8.
Ein Beispiel für die Anwendung der geänderten 3' RACE ist die jüngste Identifizierung eines Romans CCND1-MRCK Fusion gen Abschrift von Mantel-Zell-Lymphom-Zell-Linien und Krebspatienten. Die 3' UTR bestand aus Sequenzen aus der CCND1 und MRCK Gene und widerspenstigen MiRNA Verordnung9war. Die zwei verschachtelte CCND1 spezifische vorwärts Primer wurden ergänzend zu der Region unmittelbar neben und hinter der CCND1 -Stopp-Codon. Obwohl ganze Transkriptom Sequenzierung zusammen mit spezifischen Bioinformatic Hilfsmittel verwendet werden kann, um gen Fusionen innerhalb der 3' UTR-10zu erkennen, können viele Labore fehlen die finanziellen Mittel oder Bioinformatic Know-how, um diese Technologie nutzen. Daher ist 3' Rennen eine Alternative zur de-Novo -Identifizierung und Validierung von Roman Fusionsgene mit der 3' UTR. In Anbetracht der drastische Anstieg der Zahl der gemeldeten Fusionsgene sowie die Transkripte durchlesen, 3' Rennen ein mächtiges Werkzeug bei der Charakterisierung von Gen-Sequenzen11,12geworden. Darüber hinaus haben Studien gezeigt, dass diese verschiedenen Sequenzen innerhalb der 3' UTR sowie die Länge der 3' UTR mRNA-Transkript Stabilität, Lokalisierung, Übersetzbarkeit und Funktion13beeinflussen kann. Zum Teil auf ein verstärktes Interesse bei der Zuordnung des Transkriptoms, gab es eine Zunahme der Anzahl der verschiedenen DNA-Polymerasen für den Einsatz im Labor entwickelt. Es ist wichtig, festzustellen, was Arten von Änderungen an die 3' Rennen Protokoll in bestellen, nutzen Sie die verfügbaren Repertoire von DNA-Polymerasen erfolgen können.
Diese Arbeit Berichte Anpassung 3' Rennen die gesamte 3' UTR zuordnen, die PAS und die 3' Ende Spaltstelle der ANKHD1 Abschrift mit Primer im Abschnitt ANKHD1 die Abschrift und zwei verschiedene DNA-Polymerasen verschachtelt.

<table>
	<tbody>
		<tr>
			<td>HeLa cells</td>
			<td>ATCC</td>
			<td>CCL-2</td>
			<td>Cervical cancer cell line.</td>
		</tr>
		<tr>
			<td>Jeko-1 cells</td>
			<td>ATCC</td>
			<td>CRL-3006</td>
			<td>Mantle cell lymphoma cell line.</td>
		</tr>
		<tr>
			<td>Granta-519 cells</td>
			<td>DSMZ</td>
			<td>ACC-342</td>
			<td>Mantle cell lymphoma cell line.</td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma Aldrich</td>
			<td>F6178</td>
			<td>Fetal bovine serum for cell culture.</td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin</td>
			<td>ThermoFisherScientific</td>
			<td>15140122</td>
			<td>Antibiotic and antimycotic.</td>
		</tr>
		<tr>
			<td>GlycoBlue</td>
			<td>Ambion</td>
			<td>AM9545</td>
			<td>Coprecipitant.</td>
		</tr>
		<tr>
			<td>DMEM</td>
			<td>ThermoFisherScientific</td>
			<td>10569044</td>
			<td>Gibco brand cell culture media with GlutaMAX.</td>
		</tr>
		<tr>
			<td>Nuclease Free-Water</td>
			<td>ThermoFisherScientific</td>
			<td>AM9938</td>
			<td>Ambion DNase and RNAse free water(non DEPC treated).</td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s Phosphate-Buffered Solution</td>
			<td>Corning</td>
			<td>21-030</td>
			<td>1X PBS.</td>
		</tr>
		<tr>
			<td>Chloroform</td>
			<td>Sigma&#160; Aldrich</td>
			<td>C7559-5VL</td>
			<td></td>
		</tr>
		<tr>
			<td>2-propanol</td>
			<td>Sigma Aldrich</td>
			<td>I9516</td>
			<td></td>
		</tr>
		<tr>
			<td>Reagent Alcohol</td>
			<td>Sigma Aldrich</td>
			<td>793175</td>
			<td>Ethanol</td>
		</tr>
		<tr>
			<td>Ethidium Bromide solution</td>
			<td>Sigma Aldrich</td>
			<td>E1510</td>
			<td></td>
		</tr>
		<tr>
			<td>TRIzol Reagent</td>
			<td>ThermoFisherScientific</td>
			<td>15596026</td>
			<td>Monophasic phenol and guanidine isothiocyanate reagent.</td>
		</tr>
		<tr>
			<td>2X Extender PCR-to-Gel Master Mix</td>
			<td>Amresco</td>
			<td>N867</td>
			<td>2X PCR-to-Gel Master Mix&#160; containing loading dye used in routine quick PCR assays and primer search.</td>
		</tr>
		<tr>
			<td>10mM dNTP</td>
			<td>Amresco</td>
			<td>N557</td>
			<td></td>
		</tr>
		<tr>
			<td>RQ1 RNase-Free DNase</td>
			<td>Promega</td>
			<td>M6101</td>
			<td>Dnase treatment kit.</td>
		</tr>
		<tr>
			<td>Gel Loading Dye Orange (6X)</td>
			<td>New England BioLabs</td>
			<td>B7022S</td>
			<td></td>
		</tr>
		<tr>
			<td>2X Phusion High-Fidelity PCR Master Mix with HF buffer</td>
			<td>ThermoFisherScientific</td>
			<td>F531S</td>
			<td>2X PCR MasterMix containing a chimeric DNA polymerase consisting of a DNA binding domain fused to a Pyrococcus-like proofreading polymerase and other reagents.</td>
		</tr>
		<tr>
			<td>PfuUltra II Fusion HS DNA polymerase</td>
			<td>Agilent Technologies</td>
			<td>600670</td>
			<td>Modified DNA Polymerase from Pyrococcus furiosus (Pfu).</td>
		</tr>
		<tr>
			<td>RevertAid RT Reverse Transcription Kit</td>
			<td>Thermo Fischer</td>
			<td>K1691</td>
			<td>Used for&#160; reverse transcription of mRNA into&#160; cDNA synthesis. Kit includes&#160; Ribolock RNAse inhibitor, RevertAid M-MuLV reverse transcriptase and other reagents listed in manuscript.</td>
		</tr>
		<tr>
			<td>Pefect size 1Kb ladder</td>
			<td>5 Prime</td>
			<td>2500360</td>
			<td>Molecular weight DNA ladder.</td>
		</tr>
		<tr>
			<td>Alpha Innotech FluorChem Q MultiImage III</td>
			<td>Alpha Innotech</td>
			<td></td>
			<td>Used to visualise ethidium bromide stained agarose gel.</td>
		</tr>
		<tr>
			<td>Low Molecular Weight Ladder</td>
			<td>New England BioLabs</td>
			<td>N3233L</td>
			<td>Molecular weight DNA ladder.</td>
		</tr>
		<tr>
			<td>Vortex Mixer</td>
			<td>MidSci</td>
			<td>VM-3200</td>
			<td></td>
		</tr>
		<tr>
			<td>Mini Centrifuge</td>
			<td>MidSci</td>
			<td>C1008-R</td>
			<td></td>
		</tr>
		<tr>
			<td>Dry Bath</td>
			<td>MidSci</td>
			<td>DB-D1</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop 2000C</td>
			<td>ThermoFisherScientific</td>
			<td>ND-2000C</td>
			<td>Spectrophotometer.</td>
		</tr>
		<tr>
			<td>Wide Mini-Sub&#160;Cell GT Horizontal Electrophoresis System</td>
			<td>BioRad</td>
			<td>1704469</td>
			<td>Electrophoresis equipment-apparatus to set up gel</td>
		</tr>
		<tr>
			<td>PowerPac Basic Power Supply</td>
			<td>BioRad</td>
			<td>1645050</td>
			<td>Power supply for gel electrophoresis.</td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Dot Scientific</td>
			<td>AGLE-500</td>
			<td></td>
		</tr>
		<tr>
			<td>Mastercycler Gradient</td>
			<td>Eppendorf</td>
			<td>950000015</td>
			<td>PCR thermocycler.</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5810 R</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5430R</td>
			<td></td>
		</tr>
		<tr>
			<td>Wizard SV Gel and PCR Fragment DNA Clean-Up System</td>
			<td>Promega</td>
			<td>A9281</td>
			<td></td>
		</tr>
		<tr>
			<td>Zero Blunt TOPO PCR Cloning Kit, with One Shot&#160; TOP10 Chemically Competent E. coli cells</td>
			<td>ThermoFisherScientific</td>
			<td>K280020</td>
			<td></td>
		</tr>
		<tr>
			<td>MyPCR Preparation Station Mystaire</td>
			<td>MidSci</td>
			<td>MY-PCR24</td>
			<td>Hood dedicated to PCR work.</td>
		</tr>
		<tr>
			<td>Hamilton SafeAire II fume hood</td>
			<td>ThermoFisherScientific</td>
			<td></td>
			<td>Fume hood.</td>
		</tr>
		<tr>
			<td>Beckman Coulter Z1&#160;Particle&#160;Counter</td>
			<td>Beckman Coulter</td>
			<td>6605698</td>
			<td>Particle counter. For counting cells before plating&#160; for RNA extraction.</td>
		</tr>
		<tr>
			<td>Applied Biosystems Sequence Scanner Software v2.0</td>
			<td>Applied Biosystems (through ThermoFisherScientific)</td>
			<td></td>
			<td>Software to analyze Sanger sequencing data.</td>
		</tr>
	</tbody>
</table>

adapting 3' rapid amplification of cdna ends to map transcripts in cancer

Reifung der eukaryontischen mRNAs beinhaltet 3' Ende Bildung, beinhaltet die Zugabe eines Poly-Schwanzes. Um die 3'-Ende eines Gens abbilden zu können, ist die traditionelle Methode der Wahl 3' schnelle Verstärkung der cDNA enden (3' Rennen). Protokolle für 3' Rennen erfordern sorgfältige Planung und Auswahl der verschachtelten Primer innerhalb der 3' untranslatierten Region (3' UTR) des Zielgens von Interesse. Mit ein paar Modifikationen kann jedoch das Protokoll verwendet werden, um die gesamte 3' UTR und Sequenzen innerhalb der offenen Leserahmen (ORF), bietet ein noch umfassenderes Bild der Beziehung zwischen dem ORF und der 3' UTR. Dies ist neben der Kennzeichnung Polyadenylation Signal (PAS), sowie der Spaltung und Polyadenylation Website von herkömmlichen 3' Rennen zur Verfügung gestellt. Erweiterte 3' Rennen erkennt ungewöhnlichen 3' wo, einschließlich gen Fusionen innerhalb der 3' UTR, und die Sequenzinformation kann verwendet werden, um vorherzusagen, potenzielle MiRNA Bindungsstellen sowie AU reichen destabilisierende Elemente, die die Stabilität der Abschrift beeinträchtigen können.

Verschachtelte Forward Primer suchen:
Das Agarosegel von Abbildung 1 zeigt zwei unterschiedliche PCR-Gelprodukte (Bahnen 1 und 2), welche das gleiche Grundierung aber verschiedene reverse Primer weiterleiten. Bahn 3 hat ein eindeutig identifizierbares PCR-Produkt und hat eine ausgeprägte vorwärts- und Grundierung. Die ideale Primer für die PCR-basierten Reaktion zu verwenden sind di...

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Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer

Anpassung der 3' endet rasche Verstärkung der CDNA Transkripte in Krebs zuordnen

Die zwei verschiedenen 3' schnelle Verstärkung der cDNA enden (3' Rennen) Protokolle beschrieben hier machen Verwendung von zwei verschiedene DNA-Polymerasen Sequenzen zuordnen, die ein Segment der offenen Leserahmen (ORF), das Stopp-Codon und die gesamte 3' UTR eine Niederschrift mit RNA enthalten aus verschiedenen Krebszelllinien gewonnen.

Maturation of eukaryotic mRNAs involves 3&#39; end formation, which involves the addition of a poly(A) tail. In order to map the 3&#39; end of a gene, the ...

The overall goal of this procedure is to determine the sequences of mRNA transcripts from the three-prime end to regions within the protein coding region by using transcript-specific nested primers in two subsequent PCRs and sequencing the purified PCR products. This method can help answer key questions in the genetic field, such as determining the polyadenylation signal, the stop codon, and the sequence of the three-prime untranslated region of the transcript. The main advantage of this technique is that it can be applied to any transcript if the sequence of the small region of the open reading frame is The implication of this technique extend toward cancer diagnosis, because it can detect tumor specific fusion genes and splice variants.We first had the idea for this method when we identified novel transcripts with previously uncharacterized, three-primed untranslated regions. To begin the protocol, thaw the phenol and guanidine isothiocyanate cell mixture on ice. Once thawed, add 100 microliters of chloroform to the microcentrifuge tube in a PCR hood.Vortex the sample for 10 to 15 seconds until the mixture is pink and opaque. Next, incubate the sample on ice for 15 minutes. After incubation, centrifuge the sample for 15 minutes at 20, 800 g and four degrees Celsius.Carefully remove the upper colorless aqueous phase and transfer it to a new 1.5 milliliter microcentrifuge tube. Then, move on to precipitation and add an equal volume of isopropanol to the sample. Add one microliter of co-precipitant to the sample to act as a carrier for the RNA and to help visualize the RNA in subsequent steps.Incubate the samples at 80 degrees Celsius for at least four hours. After incubation, transfer the sample to a cooled centrifuge and spin for 35 minutes at 20, 800 g and four degrees Celsius. After the spin, the RNA will appear as a blue spec on the bottom of the tube.Carefully remove the isopropanol from the sample. Add 500 microliters of 80%ethanol and resuspend the pellet by briefly vortexing for three seconds. Centrifuge the sample again.Remove all the ethanol from the sample and let the sample air dry for about 10 minutes. Once the sample is dry, resuspend it in 35 microliters of RNase-free water. Then, place the sample on a heat block.Set at 65 degrees Celsius for five minutes to ensure that the RNA is fully in solution. After heating, immediately place the tube on ice. For a final reaction volume of 50 microliters, add four micrograms of total RNA with added water to a total volume of 22 microliters to a new tube.Add two microliters of the Oligo dT 25 primer from the 10 micromolar primer solution. From the reverse transcription kit, add two microliters of the RNase inhibitor, eight microliters of the 5x reaction buffer, four microliters of 10 millimolar dNTPs, and two microliters of the reverse transcriptase. Set up a reaction without reverse transcriptase to act as a negative control.Incubate the tube at 42 degrees Celsius for one hour. After incubation, transfer the tube directly to ice before transporting it to the heating block. Then, heat the tube at 75 degrees Celsius for five minutes.After heating it 75 degrees Celsius, place the tube on ice. Transfer two microliters of the cDNA to a fresh 0.5 milliliter PCR tube. Add one microliter of the forward primer and one microliter of the reverse primer from a 10 millimolar primer solution.Make up to 12.5 microliters by adding 8.5 microliters of nuclease-free water and add 12.5 microliters of the 2x PCR to gel master mix. Then, enter the PCR thermal cycling conditions listed on the screen and run the PCR. After the PCR, prepare a 1%agarose gel stained with ethidium bromide.Dissolve one gram of agarose in 100 milliliters of triacetate or TAE buffer in a 250 milliliter flask. Boil the contents in a microwave. Allow the gel to cool for one minute, and add 7.5 microliters of ethidium bromide solution in a fume hood.Mix well by swirling the flask. Pour the contents into a horizontal gel apparatus and allow the gel to solidify at room temperature in the fume hood for at least 30 minutes. Cover the gel with 1x TAE buffer and load 10 microliters of the PCR product onto the agarose gel together with three to five microliters of the DNA molecular weight ladder.Run the gel at 175 volts for 10 minutes. For the first PCR, transfer one microliter of cDNA to a PCR tube and add five microliters of 10x Pfu reaction buffer. Add one microliter of the first nested forward primer, one microliter of the T7 Oligo dT 25 primer, and one microliter of dNTPs from a 10 millimolar solution.Make up to 49 microliters of total volume by adding 40 microliters of water. Add one microliter of Pfu DNA polymerase and mix well. Then, run the PCR.Next, prepare the second PCR. Transfer two microliters of the products from the first PCR to a new PCR tube and mix it with five microliters of 10x Pfu ultra two reaction buffer. Add one microliter of the second nested PCR primer, one microliter of the T7 primer and one microliter of dNTPs.Make up to 49 microliters total reaction volume by adding 39 microliters of water and add one microliter of Pfu DNA polymerase. Run the PCR using the same PCR profile as the first PCR set up. Alternatively, instead of using Pfu DNA polymerase, a chimeric DNA polymerase can be used in the two different PCRs.Take five to ten microliters of the second PCR product and run it on an agarose gel. Finally, visualize the result on an imager. This agarose gel shows two distinct PCR products which use the same forward primer but different reverse primers and a distinct PCR product that has a distinct forward and reverse primer.The ideal primers to use for the PCR based reaction are those that give one distinct PCR product. Products from the second PCR reaction with Pfu DNA polymerase and chimeric DNA polymerase produced the same sized PCR products despite having different PCR cycling conditions for the three-prime RACE. The reverse transcriptase negative controls showed no genomic contamination of the RNA used in cDNA synthesis as well as in subsequent downstream reactions.Gel-purified PCR products were sent for sequencing. A representative Sanger sequence from a sequence chromatogram identified the location of the stop codon, putative polyadenylation signal and the site as well as the poly A tail in the three-prime RACE product. While attempting this procedure, it's important to remember to wear protective gear and gloves at all time.In addition, use sterile DNase and RNase for your reagents tubes and other disposables. Don't forget that working with phenol and chloroform can be extremely hazardous. And precautions such as using the reagents in a hood and disposing material in a designated waste container should always be taken while performing this procedure.

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