Name: Vergelijkende RNA-structuuranalyse van ontluikende en rijpe transcripten in Saccharomyces cerevisiae (Video) | JoVE Journal
Uploaded: 2026-02-27T13:20:00.000+00:00
Duration: 9 min 12 s
Description: Leer CoSTseq voor RNA-structuuranalyse van ontluikende transcripten in Saccharomyces cerevisiae. Deze computationele methode brengt RNA-polymerase-positie en basenparing gelijktijdig in kaart.

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<table><tbody><tr><td>10mM ATP</td><td>Invitrogen</td><td>18330019</td><td></td></tr><tr><td>10mM Biotin-11-CTP</td><td>Jena biosciences</td><td>NU-831-BIOX</td><td></td></tr><tr><td>10mM GTP</td><td>Invitrogen</td><td>18332015</td><td></td></tr><tr><td>10mM UTP</td><td>Invitrogen</td><td>18333013</td><td></td></tr><tr><td>10X NEBuffer 1</td><td>New England Biolabs</td><td>B7001S</td><td>Gebruikt in stap 7.2.1</td></tr><tr><td>10X Phosphate Buffered Saline (PBS)</td><td>Gibco</td><td>70011-044</td><td></td></tr><tr><td>20% SDS</td><td>RPI&nbsp;</td><td>L23100-500</td><td></td></tr><tr><td>Acid phenol:chloroform&nbsp;</td><td>Ambion</td><td>AM9722</td><td></td></tr><tr><td>AMPure XP beads voor maatselectie</td><td>Beckman Coulter&nbsp;</td><td>A63880</td><td>Gebruikt voor maatselectie in stap 7.5.1</td></tr><tr><td>Bacto Peptone</td><td>Gibco</td><td>211677</td><td></td></tr><tr><td>Bacto Yeast Extract</td><td>Gibco</td><td>212750</td><td></td></tr><tr><td>Bicine</td><td>Sigma Aldrich</td><td>B3876-100G</td><td></td></tr><tr><td>Chloroform</td><td>Sigma Aldrich</td><td>319988</td><td></td></tr><tr><td>D-(+)-GLUCOSE</td><td>Sigma Aldrich</td><td>G5767-500G</td><td></td></tr><tr><td>DIFCO AGAR</td><td>BD DIFCO</td><td>214010</td><td></td></tr><tr><td>Dimethyl Sulfate</td><td>Sigma Aldrich</td><td>D186309-5ML</td><td></td></tr><tr><td>Dynabeads&trade; MyOne Streptavidin C1 beads&nbsp;</td><td>Invitrogen</td><td>65001</td><td>Gebruikt voor Biotin Pulldown in Sectie 4.1</td></tr><tr><td>Dynabeads&trade; mRNA DIRECT&trade; Purification Kit</td><td>Invitrogen</td><td>61011</td><td>Gebruikt voor Poly A selectie in Sectie 5.1</td></tr><tr><td>EDTA, pH 8, 0.5M</td><td>Sigma Aldrich&nbsp;</td><td>03690-100ML</td><td></td></tr><tr><td>Ethanol</td><td>Sigma Aldrich</td><td>E7023-500ML</td><td></td></tr><tr><td>GlycoBlue</td><td>Invitrogen</td><td>AM9516</td><td>Co-precipitant gebruikt in stap 4.2.7 en 5.2.4</td></tr><tr><td>Induro&reg;&nbsp;Reverse Transcriptase</td><td>New England Biolabs</td><td>M0681S</td><td>RT-enzym gebruikt in stap 6.3.1</td></tr><tr><td>Isoamyl alcohol</td><td>Sigma Aldrich&nbsp;</td><td>W205710-1KG-K</td><td></td></tr><tr><td>Isopropanol</td><td>JT-BAKER</td><td>9084-05-01</td><td></td></tr><tr><td>KAPA HiFi HotStart PCR Kit&nbsp;&nbsp;</td><td>Roche</td><td>07958889001</td><td>High-fidelity DNA Pol gebruikt in 7.3.2 en 7.4.1 voor PCR-reacties</td></tr><tr><td>Magnesium Chloride</td><td>Sigma Aldrich</td><td>SLCM2154</td><td></td></tr><tr><td>MinElute PCR Purification Kit</td><td>Qiagen</td><td>28004</td><td>Gebruikt voor DNA-opruiming in stap 6.3.3 en 7.2.2</td></tr><tr><td>Mth RNA Ligase</td><td>New England Biolabs</td><td>M2611A</td><td></td></tr><tr><td>Oligo Clean &amp; Concentrator</td><td>Zymo Research</td><td>D4060</td><td>Aanbevolen voor DNA Oligo-opruiming in stap 7.1.2</td></tr><tr><td>Potassium Acetate&nbsp;</td><td>Sigma Aldrich</td><td>236497-500G</td><td></td></tr><tr><td>Potassium Chloride</td><td>JT Baker</td><td>3040-01</td><td></td></tr><tr><td>Potassium Hydroxide</td><td>Avantor</td><td>6984-04-01</td><td></td></tr><tr><td>RNA Clean &amp; Concentrator-5</td><td>Zymo Research</td><td>R1014</td><td>RNA-opruimingskit gebruikt in stap 5.3.2 na PNK-behandeling</td></tr><tr><td>RNaseOUT&trade; Recombinant Ribonuclease Inhibitor</td><td>Thermo Fisher Scientific</td><td>10777019</td><td>RNase-remmer gebruikt in stap 5.3.1 tijdens PNK-behandeling</td></tr><tr><td>Sarkosyl</td><td>IBI Scientific</td><td>IB07080</td><td></td></tr><tr><td>Sodium Acetate</td><td>Quality Biological</td><td>351-035-721</td><td></td></tr><tr><td>Sodium Chloride</td><td>Sigma Aldrich</td><td>S5150-1L</td><td></td></tr><tr><td>Sodium Hydroxide&nbsp;</td><td>Macron</td><td>7708-10</td><td></td></tr><tr><td>SUPERase&middot;In&trade; RNase Inhibitor (20 U/&mu;L)</td><td>Thermo Fisher Scientific</td><td>AM2696</td><td>RNase-remmer gebruikt in stap 6.3.1</td></tr><tr><td>T4 Polynucleotide Kinase</td><td>New England Biolabs</td><td>M0201S</td><td></td></tr><tr><td>Thermostable 5&rsquo; App DNA/RNA Ligase</td><td>New England Biolabs</td><td>M0319L</td><td></td></tr><tr><td>Tris-HCl, pH 7.4, 1M</td><td>Thermo Scientific</td><td>J60202.K2</td><td></td></tr><tr><td>Triton X-100</td><td>TEKNOVA</td><td>T1105</td><td></td></tr><tr><td>TRIzol&trade; Reagent</td><td>Invitrogen</td><td>15596-026</td><td>Voor nascent RNA-elutie in Sectie 4.2; ook vermeld als "RNA-reagens" in Sectie 4</td></tr><tr><td>&beta;-mercaptoethanol</td><td>Sigma Aldrich</td><td>M6250-1L</td><td></td></tr></tbody></table>

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Comparative RNA Structure Analysis of Nascent and Mature Transcripts in Saccharomyces cerevisiae

Vergelijkende RNA-structuuranalyse van ontluikende en rijpe transcripten in Saccharomyces cerevisiae

During transcription, nascent RNA starts base pairing as it exits RNA polymerases (Pols). This base pairing permits the formation of RNA structures that ...

comparative-rna-structure-analysis-nascent-mature-transcripts

Research

JoVE Journal

Biochemistry

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Het protocol hier beschreven is gebaseerd op het genoom-brede kwantificeren van nieuw samengestelde mRNA gezuiverd van gistcellen aangeduid met 4-thiouracil. Deze methode maakt het mogelijk om het meten van de mRNA-synthese rekken van mRNA verval en biedt dus een nauwkeurige meting van RNA polymerase II transcriptie.

Saccharomyces cerevisiae Metabole labelen met 4-thiouracil en de kwantificering van gesynthetiseerd mRNA nieuw als een Proxy voor RNA Polymerase II activiteit

Hier presenteren we een protocol voor DNAzyme-afhankelijke splijting van RNA. Dit maakt een snelle en site-afhankelijke analyse van RNA 2 '-O-Methylation mogelijk. Deze aanpak kan worden gebruikt voor de voorlopige of belangrijke beoordeling van de activiteit van snoRNA.

DNAzyme-afhankelijke analyse van rRNA 2 '-O-methylatie

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Geoptimaliseerde kwantitatieve beoordeling van de stabiliteit van enhancer-RNA in muisembryonale stamcellen

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Leer CoSTseq voor RNA-structuuranalyse van ontluikende transcripten in Saccharomyces cerevisiae. Deze computationele methode brengt RNA-polymerase-positie en basenparing gelijktijdig in kaart.

Vergelijkende RNA-structuuranalyse van ontluikende en rijpe transcripten in Saccharomyces cerevisiae

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