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

Vorremmo riconoscere Bettine Gibbs per il suo aiuto tecnico.

Indossare un camice da laboratorio, guanti e occhiali di sicurezza in ogni momento durante l'esecuzione di tutte le procedure in questo protocollo. Assicurarsi che i contenitori/provette contenenti il reagente di fenolo e guanidina isotiocianato sono aperti solo in un cappuccio certificata e smaltire i rifiuti di fenolo in un contenitore designato. Utilizzare i reagenti, i suggerimenti e privo di DNasi e RNasi provette sterili.
1. coltura cellulare
<ol><li>Crescere la linea delle cellule HeL...

<ol>
	<li>Mandel, C., Bai, Y., Tong, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+factors+in+pre-mRNA+3'-end+processing.">Protein factors in pre-mRNA 3'-end processing.</a> Cell Mol Life Sci. 65 (7-8), 1099-1122 (2008).</li><li>Danckwardt, S., Hentze, M., Kulozik, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=3'+end+mRNA+processing:+Molecular+mechanisms+and+implications+for+health+and+disease.">3' end mRNA processing: Molecular mechanisms and implications for health and disease.</a> EMBO J. 27 (3), 482-498 (2008).</li><li>Derti, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+quantitative+atlas+of+polyadenylation+in+five+mammals.">A quantitative atlas of polyadenylation in five mammals.</a> Genome Res. 22 (6), 1173-1183 (2012).</li><li>Sambrook, J., Russell, 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=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. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CFIm25+regulates+glutaminase+alternative+terminal+exon+definition+to+modulate+miR-23+function.">CFIm25 regulates glutaminase alternative terminal exon definition to modulate miR-23 function.</a> RNA. 22 (6), 830-838 (2016).</li><li>Rodu, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Molecular+Biology+in+Medicine:+The+polymerase+chain+reaction:+the+revolution+within.">Molecular Biology in Medicine: The polymerase chain reaction: the revolution within.</a> Am J Med Sci. 299 (3), 210-216 (1990).</li><li>Bertioli, D., White, B. 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>

Nonostante l'avvento delle tecnologie di sequenziamento massivo parallelo, su una base di gene di gene, 3' gara rimane ancora il metodo più facile ed economico per identificare la PAS e nucleotidi adiacenti per la coda di poli (a). L'adattamento qui descritto si espande con 3' gara per amplificare sia mappa sequenze che includono una parte di ORF, il codone di arresto e l'intero regione 3' UTR della trascrizione del mRNA di ANKHD1 . Dei principali vantaggi di 3' gara è che con pochi piccoli adattamenti, prodot...

La formazione dell'estremità 3' è un passo fondamentale nella maturazione di mRNA che comprende il clivaggio del pre-mRNA a valle di un PAS seguita dall'aggiunta di ~ 250 tipo adenine, che compongono la coda di poli (a)1,2. La proteina legante il poli (a) (PABP) si lega alla coda di poli (a), e questo protegge la trascrizione di mRNA da degradazione e facilita la traduzione1.
Le stime attuali suggeriscono che il 70% dei geni umani hanno passaggio multiplo e quindi sono sottoposti ad poliadenilazione alternativa, con conseguente più 3' estremità3. Pertanto, è importante identificare dove la coda di poli (a) si attacca al resto del 3' UTR, nonché a identificare il PAS utilizzato da qualsiasi trascrizione dato. L'avvento di sequenziatori di ultima generazione ha provocato l'identificazione simultanea del 3' UTR e il passaggio di migliaia di geni. Questo aumento della capacità di sequenziamento ha richiesto lo sviluppo di algoritmi bioinformatici per analizzare i dati che coinvolgono poliadenilazione alternativa dell'estremità 3'. Per l'individuazione del de novo o la convalida della PAS e quindi mapping dell'estremità 3' dei singoli geni da dati di sequenziamento su larga scala, 3' gara rimane il metodo di scelta4,5. Le sequenze incluse nei prodotti di cDNA di 3' gara normalmente includono solo una parte del 3' UTR che contiene la coda di poli (a), il luogo di fenditura, la PAS e le sequenze a Monte della PAS. A differenza di PCR, che richiede la progettazione e la scelta del gene specifico primer forward e reverse, 3' gara richiede solo due primers gene nidificati specifici in avanti. Quindi, la PCR richiede una conoscenza più dettagliata della sequenza del nucleotide di una grande regione del gene di essere amplificato4,6. Dato che 3' gara utilizza lo stesso reverse primer che obiettivi il poli (a) di coda per tutte le trascrizioni di poliadenilazione RNA, solo i primer in avanti devono essere gene specifico, così, solo che richiedono la conoscenza di una regione significativamente più piccola del mRNA. In questo modo l'amplificazione delle regioni in cui le sequenze non sono completamente caratterizzato4,7. Questo ha permesso 3' gara per essere utilizzato non solo per determinare l'estremità 3' del gene, ma a anche determinare e caratterizzare grandi regioni a Monte della PAS che formano una parte significativa del 3' UTR. Combinando 5' corsa con modificate 3' gara che comprende porzioni più grandi di 3' UTR e regioni fiancheggianti, è possibile completamente di sequenza o clonare un'intero trascrizione di mRNA dall'estremità 5' alla sua 3' estremità8.
Un esempio di questa applicazione di modificate 3' gara è la recente identificazione di un romanzo di trascrizione del gene CCND1-MRCK fusione da linee cellulari di linfoma a cellule mantellari e malati di cancro. 3' UTR ha consistito di sequenze di geni MRCK sia il CCND1 ed era ricalcitrante a miRNA regolamento9. I due nidificati CCND1 avanti primers specifici erano complementari alla regione immediatamente adiacente e a valle del codone di arresto di CCND1 . Anche se l'intero trascrittoma sequenziazione insieme strumenti bioinformatici specifico può essere utilizzato per rilevare le fusioni del gene entro i 3' UTR10, molti laboratori potrebbero mancare le risorse finanziarie o perizia di bioinformatica per fare uso di questa tecnologia. Quindi, 3' gara è un'alternativa per de novo identificazione e validazione di geni di fusione romanzo che coinvolge il regione 3' UTR. Considerando il drastico aumento del numero di geni di fusione segnalati nonché a leggere attraverso le trascrizioni, 3' gara è diventata un potente strumento nella caratterizzazione genica sequenze11,12. Inoltre, recenti studi hanno dimostrato che diverse sequenze all'interno di 3' UTR, nonché la lunghezza del 3' UTR può influenzare la stabilità di trascrizione mRNA, la localizzazione, la traducibilità e funzione13. Dovuto in parte a un crescente interesse nella mappatura del transcriptome, c'è stato un aumento del numero di differenti polimerasi del DNA in fase di sviluppo per l'utilizzo in laboratorio. È importante determinare quali tipi di modifiche possono essere reso a 3' protocollo di gara in ordine di utilizzare il repertorio disponibile di polimerasi del DNA.
Questo lavoro rapporti adattandosi 3' gara per mappare l'intero regione 3' UTR, la PAS e il sito di clivaggio estremità 3' della trascrizione ANKHD1 utilizzando nidificati Primer all'interno della sezione di ANKHD1 della trascrizione e due diversi DNA polimerasi.

<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

Maturazione degli mRNA eucariotico coinvolge 3' fine formazione, che prevede l'aggiunta di una coda di poli (a). Al fine di mappare il 3' estremità di un gene, il tradizionale metodo di scelta è 3' amplificazione veloce delle estremità del cDNA (3' gara). Protocolli per 3' gara richiedono l'attenta progettazione e selezione degli iniettori annidati all'interno di regione 3' non tradotta (3' UTR) del gene target di interesse. Tuttavia, con alcune modifiche il protocollo può essere utilizzato per includere l'intera regione 3' UTR e sequenze all'interno l'open reading frame (ORF), fornendo un quadro più completo del rapporto tra l'ORF e 3' UTR. Ciò è oltre alla identificazione del segnale poliadenilazione (PAS), così come il sito di clivaggio e poliadenilazione fornito dai convenzionali 3' gara. Espanso 3' gara in grado di rilevare insolito 3' UTR, tra cui fusioni del gene all'interno di 3' UTR, e le informazioni di sequenza possono essere utilizzate per prevedere i potenziali siti di legame di miRNA così come AU ricca destabilizzare gli elementi che possono influenzare la stabilità della trascrizione.

Ricerca nidificata Forward Primer:
Il gel di agarosio da La figura 1 Mostra due distinti prodotti di gel PCR (corsie 1 e 2) che utilizzano lo stesso forward primer ma diversi iniettori d'inversione. Lane 3 ha un prodotto PCR distinto e ha un distinto primer forward e reverse. I primer ideali da utilizzare per la reazione di PCR di base sono quelli che danno un prodotto di PCR distinto ...

Watch this Scientific Journal Video about Adapting 3' Rapid Amplification of CDNA Ends to Map Transcripts in Cancer at JoVE.com

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

Adattamento 3' amplificazione veloce di CDNA finisce per mappare le trascrizioni in cancro

I due diversi 3' amplificazione veloce delle estremità del cDNA (3' gara) protocolli descritto qui fanno uso di due diversi DNA polimerasi per mappare sequenze che includono un segmento dell'open reading frame (ORF), il codone di arresto e l'intero regione 3' UTR di una trascrizione usando RNA ottenute da linee cellulari tumorali differenti.

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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