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

Vi ønsker å erkjenne Bettine Gibbs for hennes teknisk hjelp.

Bruk en labfrakk, hansker og vernebriller når du utfører alle prosedyrer i denne protokollen. Kontroller at beholdere/rør med fenol og guanidine isothiocyanate reagens bare åpnes i en sertifisert hette, og kast fenol avfall i en egen beholder. Bruk DNAse RNAse-fri sterilt rør, tips og reagenser.
1. celle kultur
<ol><li>Vokse HeLa celle linjen og to suspensjon mantelen celle lymfom cellelinjer, Granta-519 og Jeko-1, i DMEM som inneholder 10% FBS og 100 U/mL Penicillin/Streptomycin i en f...

<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>

Til tross for bruk av massiv parallelle sekvensering teknologi på gene-av-genet basis, fortsatt 3' rase den enkleste og mest økonomiske metoden å identifisere PAS og nukleotider tilstøtende poly(A) halen. Tilpasningen beskrevet her utvider bruker 3' rase både forsterke og tilordne sekvenser som inkluderer en del av ORF, stopp codon og det hele 3' UTR av ANKHD1 mRNA transkripsjon. En stor fordel med 3' rase er at med noen mindre tilpasninger, kan produkter fra 3' rase klones i andre vektorer å lette nedstr?...

Dannelsen av 3 slutten er en avgjørende skritt i mRNA holdbarhet som består i spalting av det pre-mRNA nedstrøms av en PAS etterfulgt av tillegg av ~ 250 untemplated adenines, som utgjør poly(A) hale1,2. Poly(A) binding protein (PABP) binder til poly(A) halen, og dette beskytter mRNA transkripsjon fra fornedrelse, og forenkler oversettelse1.
Aktuelle anslår foreslå at 70% av menneskets gener har flere PASs, og dermed gjennomgå alternative polyadenylation, som resulterer i flere 3 ender3. Derfor er det viktig å identifisere der poly(A) halen festes til resten av de 3' UTR, i tillegg til å identifisere PAS brukes av noen gitt transkripsjon. Bruk av neste generasjons sekvensering har resultert i samtidige identifikasjon av 3' UTRs og PASs av gener. Denne økningen i sekvensering evnen har krevd utvikling av bioinformatic algoritmer å analysere data med alternative polyadenylation 3 slutten. For de novo søket eller validering av PAS og dermed tilordning av 3 slutten av enkelte gener fra storskala sekvensering data, forblir 3' rase metoden for valg4,5. Sekvenser med i cDNA produkter av 3' rase vanligvis inkluderer bare en del av de 3' UTR som inneholder poly(A) halen, webområdet cleavage, PAS og sekvenser oppstrøms av PAS. I motsetning til PCR, som krever design og bruk av genet bestemt forover og bakover grunning, krever 3' RACE bare to genet bestemt nestede frem primere. Derfor krever PCR mer detaljert kunnskap om nukleotid sekvensen av et stort område av genet blir forsterket4,6. Siden 3' rase bruker omvendt samme primer at mål poly(A) hale for alle polyadenylated RNA transkripsjoner, bare videresende primerne må være genet spesifikke, dermed kun krever kunnskap om en betydelig mindre region av mRNA. Dette gjør forsterkningen på områder som sekvensene ikke er fullt preget4,7. Dette har tillatt 3' rase ikke bare å finne 3 slutten av et gen, men å også bestemme og karakterisere store regioner oppstrøms PAS som utgjør en betydelig del av de 3' UTR. Ved å kombinere 5' løp med den endrede 3' rase som inkluderer større deler av de 3' UTR og flankemanøveren regioner, er det mulig å fullstendig rekkefølge eller klone en hele mRNA utskrift fra 5' slutten til sin 3 slutten8.
Et eksempel på dette programmet av endrede 3' rase er siste identifikasjon av en roman CCND1-MRCK fusion genet utskrift fra mantelen celle lymfom cellelinjer og pasienter. De 3' UTR besto av sekvenser fra både CCND1 og MRCK gener og var motvillig til miRNA regulering9. To nestede CCND1 bestemt frem primerne var komplementær til regionen umiddelbart tilstøtende og nedstrøms CCND1 stopp codon. Selv om hele transcriptome sekvensering med bestemte bioinformatic verktøy kan brukes til å oppdage genet fusjoner i de 3 UTR10, mange laboratorier kan mangle den økonomiske ressurser eller bioinformatic kompetanse til å gjøre bruk av denne teknologien. Derfor er 3' RACE et alternativ for de novo identifikasjon og validering av romanen fusion gener som involverer den 3' UTR. Vurderer drastisk økning i antallet rapporterte fusion gener som lese gjennom transkripsjoner, har 3' rase blitt et kraftig verktøy i karakteriserer genet sekvenser11,12. I tillegg har studier vist at forskjellige sekvenser i de 3' UTR samt lengden på den 3' UTR kan påvirke mRNA transkripsjon stabilitet, lokalisering, translatability og funksjonen13. Delvis på grunn av en økt interesse i kartlegging av transcriptome, har det vært en økning i antall forskjellige DNA polymerases utviklet for bruk i laboratoriet. Det er viktig å finne ut hva endringstypene kan gjøres til 3' rase protokoll i å utnytte det tilgjengelige repertoaret av DNA polymerases.
Dette arbeidet rapporter tilpasse 3' rase å kartlegge den hele 3' UTR, PAS og 3 slutten cleavage området av ANKHD1 transkripsjon ved hjelp av nestede primere i delen ANKHD1 transkripsjon og to forskjellige DNA polymerases.

<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

Modning av eukaryote mRNAs innebærer 3 slutten formasjon, som involverer tillegg av en poly(A) hale. For å tilordne 3 slutten av et gen, er den tradisjonelle metoden valgfrihet 3 raske forsterkning av cDNA ender (3' RACE). Protokoller for 3' rase krever forsiktig design og utvalg av nestede primere i 3 uoversatt regionen (3' UTR) i målet genet av interesse. Men med noen modifikasjoner kan protokollen brukes å inkludere hele 3' UTR og sekvenser fra åpent lesing rammen (ORF), gir en mer helhetlig bilde av forholdet mellom ORF og de 3' UTR. Dette er i tillegg til identifikasjonen av polyadenylation (PAS), samt webområdet cleavage og polyadenylation levert av konvensjonelle 3' rase. Utvidet 3' rase kanne merker uvanlig 3' UTRs, inkludert gene fusjoner i de 3' UTR, og rekkefølgen informasjonen kan brukes til å forutsi mulige miRNA bindende områder samt AU rik destabiliserende elementer som kan påvirke stabiliteten i transkripsjon.

Nestede frem Primer søk:
Agarose gel fra Figur 1 viser to ulike PCR gel produkter (baner 1 og 2) som Bruk samme Videresend primer men forskjellige omvendt primere. Lane 3 har en distinkt PCR-produkt og en tydelig forover og bakover grunning. Ideell primerne bruke for PCR basert reaksjonen er de som gir en tydelig PCR produktet (Lane 3). Fremover primer brukes i baner 1 og 2 gir sterke...

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

Tilpasse 3 slutter raske forsterkning av CDNA å tilordne utskrifter i kreft

To forskjellige 3' rask forsterkningen på cDNA ender (3' RACE) protokoller beskrevet her gjør bruk av to forskjellige DNA polymerases tilordne sekvenser som inkluderer et segment åpent lesing (ORF), stopp codon, og det hele 3' UTR av en utskrift ved hjelp av RNA Hentet fra ulike kreftcelle linjer.

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.

Research

JoVE Journal

Genetics

Merging Absolute and Relative Quantitative PCR Data to Quantify STAT3 Splice Variant Transcripts

Sammenslåing absolutt og relativ Kvantitativ PCR data for å kvantifisere Stat3 spleisevariant Avskrifter

Human signal transducer and activator of transcription 3 (STAT3) is one of many genes containing a tandem splicing site. Alternative donor splice sites 3 ...

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

Ingeniør Kunstige Faktorer å Spesifikt Manipulere alternativ spleising i humane celler

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

High Resolution Fluorescent In Situ Hybridization in Drosophila Embryos and Tissues Using Tyramide Signal Amplification

Høy oppløsning fluorescerende In Situ hybridisering i Drosophila embryoer og vev med Tyramide Signal forsterkning

In our efforts to determine the patterns of expression and subcellular localization of Drosophila RNAs on a genome-wide basis, and in a variety of ...

CRISPR-Mediated Reorganization of Chromatin Loop Structure

CRISPR-mediert reorganisering av Chromatin Loop struktur

Recent studies have clearly shown that long-range, three-dimensional chromatin looping interactions play a significant role in the regulation of gene ...

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects (TEdeff) in Cancers

A Murine Cell Line Based Model of Chronic CDK9 Inhibition to Study Widespread Non-Genetic Transcriptional Elongation Defects TEdeff in Cancers

En murine Cell line basert modell av kronisk CDK9 hemming å studere utbredt ikke-genetisk Transcriptional forlengelse defekter (TEdefinitivt) i kreft

We have previously reported that a subset of cancers is defined by global transcriptional deregulations with widespread deficiencies in mRNA transcription ...

Discrimintion and Mapping of the Primary and Processed Transcripts in Maize Mitochondrion Using a Circular RT-PCR-based Strategy

Discrimintion og kartlegging av primær og behandlet transkripsjoner i mais mitokondrie bruke en sirkulær RT-PCR-basert strategi

In plant mitochondria, some steady-state transcripts have 5&#39; triphosphate derived from transcription initiation (primary transcripts), while the ...

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Investigation of the Transcriptional Role of a RUNX1 Intronic Silencer by CRISPR/Cas9 Ribonucleoprotein in Acute Myeloid Leukemia Cells

Gransking av Transcriptional rolle en RUNX1 Intronic lyddemper av CRISPR/Cas9 Ribonucleoprotein i akutt myelogen leukemi celler

The bulk of the human genome (~98%) is comprised of non-coding sequences. Cis-regulatory elements (CREs) are non-coding DNA sequences that contain binding ...

Detection of Human Immunodeficiency Virus Type 1 (HIV-1) Antisense Protein (ASP) RNA Transcripts in Patients by Strand-Specific RT-PCR

Detection of Human Immunodeficiency Virus Type 1 HIV-1 Antisense Protein ASP RNA Transcripts in Patients by Strand-Specific RT-PCR

Påvisning av humant immunsvikt virus type 1 (HIV-1) Antisens protein (ASP) RNA transkripsjoner hos pasienter med strand-spesifikk RT-PCR

In retroviruses, antisense transcription has been described in both human immunodeficiency virus type 1 (HIV-1) and human T-lymphotropic virus 1 (HTLV-1). ...

Efficient Transcriptionally Controlled Plasmid Expression System for Investigation of the Stability of mRNA Transcripts in Primary Alveolar Epithelial Cells

Effektivt transkripsjonskontrollert plasmid uttrykkssystem for undersøkelse av stabiliteten av mRNA transkripsjoner i primær alveorisk epitelceller

Studying posttranscriptional regulation is fundamental to understanding the modulation of a given messenger RNA (mRNA) and its impact on cell homeostasis ...

Screening Sperm for the Rapid Isolation of Germline Edits in Zebrafish

Screening sperm for rask isolering av germline redigeringer i sebrafisk

The advent of targeted CRISPR-Cas nuclease technologies has revolutionized the ability to perform precise genome editing in both established and emerging ...