Name: single cell analysis of transcriptionally active alleles by single molecule fish
Uploaded: 2020-09-20T14:00:00
Description: Watch this Scientific Journal Video about Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH at JoVE.com

MAM, FS og MGM er finansiert av NIEHS (Prosjekt 4, P42ES027704, PI, Rusyn). MAM, FS, RMM, MGM og PKS støttes av CPRIT-finansiert GCC Center for Advanced Microscopy and Image Informatics (RP170719). Imaging støttes også av Integrated Microscopy Core ved Baylor College of Medicine med finansiering fra NIH (DK56338 og CA125123), CPRIT (RP150578), Dan L. Duncan Comprehensive Cancer Center og John S. Dunn Gulf Coast Consortium for Chemical Genomics.

For å sikre optimale resultater krever den våte labdelen av denne protokollen standard RNase-frie forholdsregler i alle trinn. For eksempel er bruk av filtrerte pipettespisser, sterile kar og RNase-frie buffere sterkt oppmuntret. Bruk av RNase-hemmere som vanadyl ribonucleoside-komplekser foreslås også, spesielt for RNAer med lavt overflodsmål.
1. Cellekultur og eksperimentelt oppsett
<ol><li>Opprettholde tilhenger MCF-7 brystkreftceller (ATCC #HTB-22) i en T75 vev kult...

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SmFISH-metoden som er beskrevet, er basert på tidligere publiserte protokoller7,12,14. I denne protokollen forklarer vi de kritiske trinnene som ble optimalisert fra våtlaboratoriet (inkludert såingstetthet, fikseringstid, permeabilisering og sondekonsentrasjon), til avbildning og bildeanalyse, og gir en full eksperimentell rørledning for laboratorier som er interessert i å utføre enkeltcelleanalyse av gentranskripsjon.</p...

Gentranskripsjon og oversettelse er de to store prosessene som er involvert i biologiens sentrale dogme, fra DNA-sekvensen til RNA til protein1. I denne studien fokuserer vi på produksjon av messenger RNA (mRNA) under transkripsjon. Det gryende RNA-molekylet inneholder både ikke-kodingsdroner og kodingseksoner. Introner er co-transkripsjonelt spleiset ut før mRNA beveger seg inn i cytoplasma for oversettelse på ribosomer2,3.
Tradisjonelt utføres målingen av mRNA i bulkpopulasjoner av celler (hundrevis til millioner) med klassiske analyser som RT-qPCR eller Northern blotting. Selv om de er svært kraftige, er disse metodene begrenset, da de ikke gir innsikt i celle-til-celle-variasjon (fenotypisk heterogenitet), identifisering av antall transkripsjonsaktive alleler, og kanskje enda viktigere, mangler romlig informasjon. Mer nylig begynte genom brede teknologier som tillater enkeltcellet RNA-sekvensering (RNAseq) å bygge bro mellom avbildningsmetoder og sekvensering4. RNAseq lider imidlertid av relativt lav deteksjonseffektivitet, og behandlingstrinnene resulterer i fullstendig tap av romlig informasjon5. Selv om enkeltcellet RNAseq kan gi innsikt i fenotypisk heterogenitet blant en populasjon av isogene celler, kan RNA fluorescens in situ hybridisering (RNA-FISH) legge til rette for en mer fullstendig utforskning av målgenuttrykk på romlig nivå, og på allel-by-allele basis6,7.
RNA FISH er en teknikk som gjør det mulig å oppdage og lokalisere mål RNA-molekyler i faste celler. I motsetning til tidligere, arbeidskrevende metoder, bruker dagens toppmoderne RNA-FISH kommersielt tilgjengelige nukleinsyresonder som er komplementære til målet RNA-sekvenser, og disse sondene hybridiserer til sine mål av Watson-Crick base parring8. De tidlige in situ hybridiseringsteknikkene involverte en lignende protokoll etablert av Gall og Pardue i 1969, da en prøve ble behandlet med nukleinsyresonder som spesielt hybridiserte til et mål RNA9. Opprinnelig ble analysen utført ved hjelp av radioaktiv eller kolorimetrisk deteksjon; Men på 1980-tallet åpnet utviklingen av fluorescens in situ hybridisering (FISH) protokoller til DNA FISH og senere RNA FISH ruten mot mer sensitiv deteksjon, og potensialet for multipleksing10,11.
Videre utvikling i RNA FISH har ført til evnen til å oppdage og kvantifisere enkle RNA-molekyler (smFISH)12,13. Direkte deteksjon er en metode der sondene selv er merket med fluoroforer. En utfordring med smFISH er at det er behov for nok hybridiserende sonder/mål til å lette påvisning av fluorescerende signaler som distinkte, diffraksjonsbegrensede flekker. For å løse dette problemet kan man bruke et sondesett med korte enkeltstrengede DNA-oligonukleotider komplementære til ulike områder av målet RNA8,12,14. Bindingen av flere sonder øker den lokale fluorofortettheten, noe som gjør RNA synlig som et tydelig, høyintensitetspunkt ved fluorescensmikroskopi. Denne metoden er fordelaktig fordi off-target binding av oligonukleotider i sondesettbassenget kan skilles fra det sanne RNA-spotsignalet ved kvantitativt å analysere spotstørrelsen og intensiteten for å skille mellom sant signal og falske oligonukleotidbinding, og dermed legge til rette for mer nøyaktig analyse ved å redusere falske positiver12.
Alternative metoder for å oppdage mRNA er den klassiske BAC klonebaserte nick oversettelsesmetoden og det mer nylig utviklede forgrenede DNA-systemet. I den BAC-klonede nick-oversettelsesmetoden er DNA-et som skal merkes enzymatisk nicked og et nytt nukleotid legges til den eksponerte 3' enden. Aktiviteten til DNA-polymerase Jeg legger til et merket nukleotid som resulterer i ønsket sonde15,16. Den forgrenede DNA-teknikken involverer oligonukleotidprober som hybridiserer i par til RNA-mål, og disse sondene forsterkes ved hjelp av flere signalforsterkningsmolekyler. Denne metoden kan forbedre signal-til-støy-forholdet betydelig, men forsterkningen kan forskyve nøyaktig kvantifisering siden fluorescerende flekker kan være vilt forskjellige i størrelse og intensitet17.
Som et modellsystem for denne protokollen, som er fullt ansatt som en del av NIEHS Superfund Research Program deltakelse for å kvantifisere effekten av endokrine forstyrrende kjemikalier i Galveston Bay / Houston Ship Channel, brukte vi østrogen reseptoren (ER) positiv brystkreft celle linje MCF-7 behandlet over natten med en ER agonist, 17β-estradiol (E2)7,18,19. E2 regulerer mange ER-målgener, inkludert det prototypiske ER-målgenet, GREB17,20,21,22. SmFISH-protokollen som er beskrevet her, bruker et sett med to spektralt separerte sondesett rettet mot GREB1; en som hybridiserer til exons og den andre til introns, noe som muliggjør måling av både moden og nascent RNA7. Vi bruker deretter epifluorescence mikroskopi kombinert med dekonvolusjon til bilde smFISH-merkede prøver, og bruker deretter bildeanalyserutiner for å kvantifisere antall aktive alleler og modne RNAer per celle.

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		<col />
	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">17&#946; Estradiol</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">E2257</td>
			<td style="width:167px;">CAS Number 50-28-2</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Ambion SSC (20X) Buffer, Rnase-free</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">AM9770</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Corning DMEM with L-Glutamine and 4.5 g/L Glucose</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT10017CV</td>
			<td>Manufactured by Invitrogen</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">DAPI</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">D8417</td>
			<td>Without sodium pyruvate</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dextran Sulfate, 50% Solution Sterile</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">3730-100ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dulbecco&#39;s Phosphate Buffered Saline Ca++/Mg++</td>
			<td style="width:71px;">Corning</td>
			<td style="width:119px;">21030CV</td>
			<td>Manufactured by Calbiochem</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ethanol, 200 Proof (100%)</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">07-678-005</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Falcon 24-Well Clear Flat Bottom TC-treated Multiwell Cell Culture Plate</td>
			<td style="width:71px;">Corning</td>
			<td align="right" style="width:119px;">353047</td>
			<td>Manufactured by Decon Laboratories</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Fetal Bovine Serum (FBS), 500 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">50-753-2978</td>
			<td>Manufactured by Gemini Bio Products</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">GE Healthcare DeltaVision LIVE High Resolution Deconvolution Microscope</td>
			<td style="width:71px;">GE Healthcare</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Hyclone Water, Molecular Biology Grade</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">SH3053802</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Immersion Oil 1.516</td>
			<td style="width:71px;">GE Healthcare Life Sciences</td>
			<td align="right" style="width:119px;">29162940</td>
			<td>Manufactured by GE Healthcare Bio-Science</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">L-glutamine Solution</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT25005Cl</td>
			<td>Manufactured by Corning</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">MCF-7 cells</td>
			<td style="width:71px;">ATCC</td>
			<td style="width:119px;">HTB-22</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Molecular Grade Formamide</td>
			<td style="width:71px;">Promega</td>
			<td style="width:119px;">PAH5051</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Olympus 60x/1.42 NA Objective</td>
			<td style="width:71px;">Olympus</td>
			<td style="width:119px;"></td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Parafilm</td>
			<td style="width:71px;">Bemis Company, Inc.</td>
			<td style="width:119px;">52858-076</td>
			<td>Distributed by VWR</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Paraformaldehyde, 16% Solution, EM Grade</td>
			<td style="width:71px;">Electron Microscopy Sciences</td>
			<td align="right" style="width:119px;">15710</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Penicilin-Streptomycin Solution</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">MT3001Cl</td>
			<td>Manufactured by Corning</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Ribonucleoside Vanadyl Complex</td>
			<td style="width:71px;">VWR</td>
			<td style="width:119px;">101229-716</td>
			<td>Manufactured by New England Biosciences</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">RNase Away</td>
			<td style="width:71px;">Ambion</td>
			<td align="right" style="width:119px;">9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium pyruvate, 100 mM</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">11-360-070</td>
			<td>Manufactured by Gibco</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Thermo Scientific Glass Coverslips, #1.5</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">12-545-81P</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Triton X-100, 100 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">BP151-100</td>
			<td>Manufactured by Fisher BioReagents</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Trypsin-EDTA Solution 0.25%</td>
			<td style="width:71px;">Sigma-Aldrich</td>
			<td style="width:119px;">T4049-500ML</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Vectashield Antifade Mounting Medium 10 mL</td>
			<td style="width:71px;">Fisher Scientific</td>
			<td style="width:119px;">NC9265087</td>
			<td>Manufactured by Vector Laboratories</td>
		</tr>
	</tbody>
</table>

single cell analysis of transcriptionally active alleles by single molecule fish

Gentranskripsjon er en viktig prosess i cellebiologi, og gjør det mulig for celler å tolke og reagere på interne og eksterne signaler. Tradisjonelle bulkpopulasjonsmetoder (Northern blot, PCR og RNAseq) som måler mRNA-nivåer, mangler evnen til å gi informasjon om celle-til-celle variasjon i svar. Presis enkeltcellet og allelisk visualisering og kvantifisering er mulig via enkeltmolekyl RNA fluorescens in situ hybridisering (smFISH). RNA-FISH utføres ved hybridisering av mål-RNAer med merkede oligonukleotidprober. Disse kan avbildes i middels/høy gjennomstrømningsmodaliteter, og gjennom bildeanalyserørledninger gir kvantitative data om både modne og gryende RNAer, alt på enkeltcellenivå. Fikserings-, permeabiliserings-, hybridiserings- og bildetrinnene er optimalisert i laboratoriet over mange år ved hjelp av modellsystemet som er beskrevet her, noe som resulterer i vellykket og robust enkeltcelleanalyse av smFISH-merking. Hovedmålet med prøveforberedelse og -behandling er å produsere bilder av høy kvalitet preget av et høyt signal-til-støy-forhold for å redusere falske positiver og gi data som er mer nøyaktige. Her presenterer vi en protokoll som beskriver rørledningen fra prøveforberedelse til dataanalyse i forbindelse med forslag og optimaliseringstrinn for å skreddersy til spesifikke prøver.

For å analysere hormonelle responser via smFISH, valgte vi for eksempel østrogenreseptormodellen (ER) som brukes i analyser med høy gjennomstrømning for å bestemme tilstedeværelsen av endokrine forstyrrende kjemikalier i miljøkatastrofer i sammenheng med deltakelse i NIEHS Superfund Research Program7. I dette eksperimentet ble tilhenger MCF-7 brystkreftceller behandlet med ER-agonisten 17β-østradiol (E2, 10 nM) eller kjøretøyet (DMSO) i 24 timer. Spektralt separerte sondesett mot GREB1 ...

Watch this Scientific Journal Video about Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH at JoVE.com

Single Cell Analysis Of Transcriptionally Active Alleles By Single Molecule FISH

Enkeltmolekyl RNA fluorescens in situ hybridisering (smFISH) er en metode for å nøyaktig kvantifisere nivåer og lokalisering av spesifikke RNAer på enkeltcellenivå. Her rapporterer vi våre validerte laboratorieprotokoller for behandling, avbildning og bildeanalyse av våtbenker for encellet kvantifisering av spesifikke RNAer.

Enkeltcelleanalyse av transkripsjonsaktive alleler av enkeltmolekyl fisk

Gene transcription is an essential process in cell biology, and allows cells to interpret and respond to internal and external cues. Traditional bulk ...

Research

JoVE Journal

Biochemistry

High Precision FRET at Single-molecule Level for Biomolecule Structure Determination

Høy presisjon FRET ved enkeltmolekylivå for biomolekylstrukturbestemmelse

A protocol on how to perform high-precision interdye distance measurements using F&#246;rster resonance energy transfer (FRET) at the single-molecule ...

Single-molecule Manipulation of G-quadruplexes by Magnetic Tweezers

Single-molekylet manipulering av G-quadruplexes av magnetiske pinsett

Non-canonical nucleic acid secondary structure G-quadruplexes (G4) are involved in diverse cellular processes, such as DNA replication, transcription, RNA ...

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Bruker tre farger Single-molekylet bånd å studere sammenhengen av Protein interaksjoner

Single-molecule F&#246;rster resonance energy transfer (smFRET) has become a widely used biophysical technique to study the dynamics of biomolecules. For ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

Kovalente immobilisering av proteiner for ett molekyl Force spektroskopi

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Proteome-wide Quantification of Labeling Homogeneity at the Single Molecule Level

Proteom hele kvantifisering av merking homogenitet på ett molekyl nivå

Cell proteomes are often characterized using electrophoresis assays, where all species of proteins in the cells are non-specifically labeled with a ...

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules

Single-molekylet Tracking mikroskopi-et verktøy for fastsettelse av Diffusive statene cytosolic molekyler

Single-molecule localization microscopy probes the position and motions of individual molecules in living cells with tens of nanometer spatial and ...

Conventional BODIPY Conjugates for Live-Cell Super-Resolution Microscopy and Single-Molecule Tracking

Konvensjonelle BODIPY-konjugater for live-cell superoppløsningsmikroskopi og enkeltmolekylsporing

Single molecule localization microscopy (SMLM) techniques overcome the optical diffraction limit of conventional fluorescence microscopy and can resolve ...

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

En In Vitro single-molecule imaging assay for analyse av cap-avhengig oversettelse kinetikk

Cap-dependent protein synthesis is the predominant translation pathway in eukaryotic cells. While various biochemical and genetic approaches have allowed ...

Single Molecule Fluorescence Energy Transfer Study of Ribosome Protein Synthesis

Enkeltmolekyl fluorescens energioverføringsstudie av ribosomet proteinsyntese

The ribosome is a large ribonucleoprotein complex that assembles proteins processively along mRNA templates. The diameter of the ribosome is approximately ...

Single-Molecule Imaging of EWS-FLI1 Condensates Assembling on DNA

Enkeltmolekylavbildning av EWS-FLI1-kondensater som monteres på DNA

The fusion genes resulting from chromosomal translocation have been found in many solid tumors or leukemia. EWS-FLI1, which belongs to the FUS/EWS/TAF15 ...