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Medicine

Transkriptom analyse af menneskets retinale kirurgiske prøver vha. Journal

Published: August 14, 2013 doi: 10.3791/50375
Automatic Translation
1,2,3,4, 1,2,3, 1,2,3, 1,2,3, 4, 1,2,3, 1,2,3
1U968, Institut National de la Santé et de la Recherche Médicale, 2UMR S 968, Université Pierre et Marie Curie, 3UMR 7210, Centre National de la Recherche Scientifique, 4Départment d'Ophtalmologie, Centre Hospitalier Universitaire de Bordeaux

Summary

Vi brugte retina prøver fra retinectomy for en transkriptom analyse af nethindeløsning. Vi udviklede en procedure, der gør det muligt for RNA bevaring mellem de kirurgiske blokke og laboratoriet. Vi standardiseret en protokol til oprensning af RNA ved cæsiumchlorid-ultracentrifugering at sikre, at de oprensede RNA'er er egnet til microarray analyse.

Abstract

Nethindeløsning (RD) beskriver en adskillelse af den neurosensoriske nethinde fra den retinale pigmenterede epithelium (RPE). RPE er afgørende for en normal funktion af de lysfølsomme neuroner, de fotoreceptorer. Nethindeløsning fra RPE skaber en fysisk hul, der er fyldt med ekstracellulære væske. RD indleder cellulære og molekylære hændelser, der påvirker både neurosensoriske nethinden og RPE siden fysiologiske udveksling af ioner og metabolitter er alvorligt foruroliget. Konsekvensen for vision er relateret til varigheden af løsrivelse da en hurtig reapposition af de to væv resulterer i genoprettelsen af synet 1.. Behandlingen af ​​RD er udelukkende kirurgisk. Fjernelse af glasagtige gel (vitrektomi) efterfølges af fjernelse ikke væsentlig del af nethinden omkring den fritliggende område at favorisere nethindeløsning. De fjernede retinale eksemplarer er res nullius (intet) og dermed normalt kasseres.At inddrive RNA fra disse kirurgiske prøver vi udviklet procedure tidsskrift, der tillader RNA bevaring under overførslen fra den kirurgiske blok til laboratoriet. Vi har også standardiseret en protokol til oprensning af RNA ved cæsiumchlorid-ultracentrifugering at sikre, at de oprensede RNA'er er egnede til globale genekspression analyse. Kvaliteten af ​​RNA blev valideret både ved RT-PCR og microarray analyse. Analyse af data viser en simultan inddragelse af inflammation og fotoreceptordegenerering løbet RD.

Introduction

Det vigtigste terapeutiske mål i nethindeløsning (RD) er at finde en måde at begrænse fotoreceptor celleskader og retinal inflammation som følge af adskillelsen af ​​fotoreceptorer fra retinale pigmenterede epitelceller. Under RD, er RPE celler aktiveres, migrere, dedifferentiate og formere ved overfladen af ​​den fritliggende nethinden, udøve kontraktile kræfter fører til komplikationer. Transcriptomics analyse af RD er en måde at identificere target gener med ændret udtryk efter RD og dermed de fremtidige terapeutiske molekyler, der kunne forbedre den endelige visuelle resultat i kombination med kirurgi. Det er velkendt ribonucleinsyre (RNA) er ikke stabil som er deoxyribonukleinsyre (DNA), sidstnævnte bliver brugt i vid udstrækning til genetiske undersøgelser, havde dens stabilitet tilladt sekventering af Neanderthal genom fra palæontologiske prøver af mere end 30.000 år gammel 2.. Transkription af DNA af generne fra genomet i messenger-RNA erstørre proces genekspression og mRNA er labil for at udgøre et signal. RNA'er er meget hurtigt nedbrudt af RNAse enzymer, der udtræden signalet. Når væv er isoleret fra en organisme, de RNA er meget ofte nedbrydes, før udtrykket er undersøgt i et forskningslaboratorium. Uregelmæssig RNA er ikke egnet til analyse genekspression. Fordi laboratoriet personale ikke kan deltage i kirurgi, vi udviklet en procedure, der er let og kræver kun, at kirurgen til at inddrive væv i en passende RNAse-fri opløsning. RNA fra vævene er stabil og kan analyseres uden nogen tegn på nedbrydning efter 72 timer ved stuetemperatur i denne opløsning. RNA'erne fra prøver oprenses ved en standardiseret fremgangsmåde der involverer en ultracentrifugering på en cæsiumchloridgradient efter at være blevet overført til laboratoriet 3.. Derefter er kvaliteten af ​​RNA'erne vurderes ved agarosegelelektroforese og med RT-PCR. RNA oprensningsprotokol harfordel at adskille molekylerne i henhold til deres densitet, der er forskellig for DNA og RNA, og sikrer, at RNA ikke er forurenet med et DNA-molekyler, der vil generere kunstig signaler i genekspressionsstudier. Desuden er de transfer RNA'er (tRNA'er), som er kvantitativt mest rigelige RNA inden for en celle, adskilles efter samme fysiske egenskab fra det ribosomale RNA (rRNA), og messenger RNA (mRNA), de to sidste er de slutprodukt af rensningsprocessen. Fjernelsen af tRNA fra præparatet er nyttigt, da de fleste af microarray analysemetoderne indebar brug af reverse transkriptase og RNA polymeraser, der inhiberes af tRNA 4-6. De rensede RNA fra kirurgiske prøver er mærket ved hjælp af standard protokol og hybridiseret til en microarray chip, og resultaterne analyseres ved hjælp af to komplementære metoder, den falske opdagelse sats metode, og ved hjælp af en ny metode baseret på gensidig information og visualiseringzed på webbaserede server Retinobase 7,8.

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Protocol

1.. Journal: procedure til inddrivelse af prøver fra Kirurgisk Block

I laboratoriet

  1. Få en indførsel kontrakt fra express rederi.
  2. Fyld 10 (eller 25) skibsfart formularer med den postadresse laboratoriet angiver kontaktperson i laboratoriet (telefonnummer og e-mailadresse).
  3. Forbered 10 (eller 25) prøver formularer nummereret fra 1 til 10 (eller 25). Disse former omfatter dedikerede rum til at informere om a) anonym identifikation af patienten, b) datoen for kirurgi og c) eventuelle supplerende bemærkninger kirurgen ønsker at tilføje. Forbered 10 (eller 25) konvolutter (150 x 210 mm).
  4. Fremstillet ved hjælp af RNAse-fri reagenser 25 (eller 65) ml 6 M guanidin-chlorid i diethylpyrocarbonat (DEPC)-behandlet H2O (GHCI).
  5. Fyld 10 eller 25 nummererede 5 ml sterile polyethylen rundbundede rør med 2,4 ml GHCI opløsning.
  6. Brugte argongas at fylde den øverste del af disse rør, end pressen tightly på hætten for at forhindre oxidation af GHCI løsning over flere års lagring i den kirurgiske kabinet.
  7. Indføre hver 5 ml rør i en 50 ml steril polypropylen konisk bund med skruelåg. Brug et stykke rent papir væv til at holde 5 ml rør i 50 ml rør, luk røret.
  8. Placer de 50 ml rør på en polystyren rack og rack i en papkasse med de konvolutter, shipping formularer, prøver formularer.
  9. Indsæt vejledningen (se: 1,12-1,19) inde i låget på papkassen for at lette deres læsning.
  10. Send papkasse med posten til kontaktpersonen på hospitalet.

På hospitalet

  1. Bringe papkassen fra den kirurgiske kabinettet til kirurgiske rum. Forsigtig Bemærk: hvis proceduren involverer en immunkompromitteret patient, bør pappet ikke bringes til kirurgiske rum.
  2. Under operationen, placeres retinal specimda i nummereret 5 ml steril polypropylen fyldt med GHCI opløsning. Lukke tæt røret.
  3. Retur røret kortvarigt.
  4. Glasset anbringes på blod rør rocker i 10 min.
  5. Udfyld den medfølgende prøve nummererede.
  6. Rapport identifikationsnummeret på den tilsvarende nummererede 50 ml rør.
  7. Indføre 5 ml rør med den kirurgiske prøve i 50 ml rør, udskifte papir væv og skru røret.
  8. Indføre røret og fyldes prøve form til det i en af ​​de ledsagende konvolutter. Lukke den.
  9. Ring til express rederi til afhentning.

2.. RNA Oprensning

I laboratoriet

  1. Homogenisere prøven i sin 5 ml rør polypropylen med GHCI løsning med en homogenisator for 1 min, mens du flytter røret op og ned.
  2. Tilføj 270 ul 2 M kaliumacetat pH 5,0. Ryst kraftigt i 10 minutter ved at placere røret på et rysteapparat i sinvandret position (420 min -1).
  3. Centrifugeres 10 minutter ved 5.000 rpm (6.500 xg) ved 20 ° C.
  4. Aspirer problemfrit den opløselige fraktion uden at forstyrre bundfaldet.
  5. Overførsel til en 14 ml sterilt rør.
  6. Tilføj 5,3 ml af 100 mM Tris pH 8,0, N-lauroylsarcosin 1%.
  7. Tilføj 3,2 g cæsiumchlorid (CsCl) og blandes røret ved hvirvelbehandling.
  8. Tilføj 1,8 ml CsCl / ethylendiamintetraeddikesyre (EDTA) i en steril 11 ml polyallomer centrifugerør.
  9. Med en 10 ml steril Pasteur pipette, RNA opløsning på 1,8 ml CsCI / EDTA ved at glide langsomt på kanten af ​​røret for at undgå at forstyrre tætheden pude.
  10. Anbring glassene (et andet rør indeholdende buffere uden nethinden hvis nødvendigt) ind i rotoren.
  11. Centrifuger 24 timer ved 32.000 rpm (225.000 xg) ved 20 ° C.
  12. Tag den overlegne del af løsningen med en steril Pasteur-pipette, og kassér den.
  13. Fjern gradvist mens du kontrollererøjeblik, hvor DNA (tyktflydende) suges med en anden steril Pasteur-pipette, og kassér den.
  14. Fjern den resterende opløsning pas på ikke at frigive RNA pellet med en tredje steril Pasteur pipette.
  15. Sektion bunden af ​​røret med en skalpel flamme-steriliseret, og derefter sætte den resterende del af røret op og ned på en steril blik.
  16. Vend glasset og skylles forsigtigt med 160 ul GHCI.
  17. Lad pellet tørre i 10 min.
  18. Pellet resuspenderes i 150 pi (10 mM Tris, pH 7,5-1 mM EDTA - 0,1% SDS).
  19. Opløsningen overføres til en 2 ml sterilt mikrocentrifugerør, og derefter høste resterende pellet med 30 pi (10 mM Tris, pH 7,5-1 mM EDTA - 0,1% SDS).
  20. Tilsæt 150 pi (10 mM Tris, pH 7,5-1 mM EDTA).
  21. Tilsæt 30 gl 3 M natriumacetat, pH 5,0, vortex røret.
  22. Tilføj 900 pi ethanol 100% (-20 ° C), vortex røret.
  23. Glasset anbringes 30 min i smeltende is.
  24. Centrifuge røret 30 minutter ved 15.000 rpm ved 4 ° C.
  25. Aspirer fint den opløselige fraktion, og kassér den.
  26. Tilsæt 500 pi 70% ethanol (RT), vortex røret.
  27. Centrifuger røret 20 minutter ved 15.000 rpm (18.000 xg) ved 4 ° C.
  28. Gentag rensningen trin (70% ethanol).
  29. Centrifuger kortvarigt og fjerne det resterende ethanol med en P200 pipette.
  30. Lad pillen lufttørre i 10 min.
  31. Pellet resuspenderes i 50 pi DEPC-behandlet H 2 O.
  32. Bland grundigt ved vortex.
  33. Inkuber 15 minutter ved 45 ° C i et vandbad.

3.. RNA Analyse ved gelelektroforese

  1. Hæld en agarosegel inde i et kemisk stinkskab.
  2. I en steril 1,5 ml mikrocentrifugerør, tilsættes 2 pi af RNA, der skal analyseres og 6,4 pi prøve prep buffer.
  3. I en anden 1,5 ml rør, tilsættes 3 pi RNA standarder og 9,6 pi prøve prep buffer.
  4. Opvarme rørene 15 minutter ved 65 ° C, Læg dem på is.
  5. Tilsæt 1 pi ethidiumbromid (EB) loadingpuffer uden farvestof i RNA-prøverøret og 1 pi EB buffer med farvestof i RNA-standarder røret.
  6. Kør gelen under 80-100 V Inde en kemisk hætte i flydende buffer, indtil en af ​​farvestofferne (bromphenolblåt) når 2/3 af bunden af ​​gelen.
  7. Skyl gelen to gange 15 minutter med 250 ml de DEPC-behandlet 2x SSC.
  8. Tag et digitaliseret billede under UV-belysning.
  9. Beregne forholdet mellem det øvre bånd (23S rRNA) og det nederste bånd (16S rRNA).

4.. Endelig Oprensning af RNA

  1. Justere lydstyrken af RNA-prøve til 100 pi med DEPC-behandlet H2O (hvis nødvendigt).
  2. Tilføj 100 ul Acid phenol (1 ml phenol mættet i DEPC-behandlet H 2 O + 130 pi 50 mM natriumacetat, pH 5,2), vortex kraftigt.
  3. Centrifugeres 10 min ved 13.000 rpm (15.000 xg) ved stuetemperatur.
  4. Recover omhyggeligt og overføre den vandige fase (øvre fase) i en roman en steril 1,5 ml mikrocentrifugerør.
  5. Tilsæt 10 gl 3 M natriumacetat, pH 5,2 og 250 pi ethanol 100% (-20 ° C).
  6. Inkuber røret 30 min i smeltende is.
  7. Centrifuger røret 30 minutter ved 14.000 rpm (18.000 xg) ved 4 ° C.
  8. Aspirer omhyggeligt den opløselige fraktion, og kassér den.
  9. Tilsæt 500 pi af 70% ethanol (stuetemperatur), og vortex røret.
  10. Centrifuger røret 20 minutter ved 14.000 rpm (18.000 xg) ved 4 ° C.
  11. Gentag rensningen trin (70% ethanol).
  12. Centrifuger kortvarigt og fjerne det resterende ethanol med en P200 pipette.
  13. Lad lufttørre pellet i 10 min.
  14. Pelleten resuspenderes i 50 pi DEPC-behandlet H 2 O.
  15. Inkuber derefter i 15 min ved 45 ° C.
  16. Absorbansen (Abs) ved 260 nm og 280 nm på 2 ul af RNA-prøve. Beregnet forholdet Abs260/Abs280.
    17. <li> Opbevar RNA-prøve ved -80 ° C (stabile i adskillige år).

5.. Løsninger og rengøringsprocedurer

  1. Kaliumacetat 2 M, pH 5,0: Opløs 19,6 g kaliumacetat i 70 ml DEPC-behandlet H 2 O. Rengør pH-meter proben ved opblødning i med 1M NaOH i 15 minutter efterfulgt af 5 skylninger med DEPC-behandlet H 2 O. Juster til pH 5,0 med eddikesyre derefter justere volumen til 100 ml med DEPC-behandlet H 2 O.
  2. Tris-HCI 100 mM, pH 8,0, N-lauroylsarcosin 1%: Opløs 2 g N-lauroylsarcosin (under en emhætte) i 40 ml 0,5 M Tris-base pH 8,0. Justere volumen til 200 ml med DEPC-behandlet H2O) CsCI / EDTA: 96 g CsCI i 50 ml 10 mM EDTA pH 7,5 fremstillet med DEPC-behandlet H 2 O. Autoklave og justere volumen til 100 ml med DEPC-behandlet H 2 O. Check for pH <7,5.
  3. Agarosegel 1%: Sæt 1 g agarose i 62 ml DEPC-behandlet H 2 O. Kog i en mikrobølgeovn. Tilføj 18 ml 12,3 Mformaldehyd, 20 ml 5x MOPS. Hæld i en kemisk hætte.
  4. Sample prep Buffer: At være forberedt ekstemporale. Bland i en kemisk hætte 8 pi 5x MOPS, 16 pi på 12,3 M formaldehyd og 40 ul af formamid.
  5. EB loading buffer (uden farve): At være forberedt ekstemporale. Tilsæt 4 gl 10 mg / ml ethidiumbromid i 20 ul 80% glycerol fremstillet med DEPC-behandlet H 2 O.
  6. EB loading buffer (med farve): At være forberedt ekstemporale. Tilsæt 2 pi 10 mg / ml ethidiumbromid i 10 pi af 80% glycerol indeholdende 0,25% xylen-cyanol, 0,25% bromphenolblåt med DEPC-behandlet H 2 O.
  7. Running buffer: Til 60 ml 5x MOPS, tilsættes 54 ml 12,3 M formaldehyd og 186 ml DEPC-behandlet H 2 O.
  8. DEPC-behandlet H2O: Inkubér destilleret vand med 0,1% v / v diethylpyrocarbonat i 2 timer ved 37 ° C under omrøring. Steriliseres ved Autoklave.
  9. Rengøring homogenisatoren: Tilføj 25 ml rengøringsmiddel (RBS) til 1 L DEPC-behandlet H2O, og immerge aksen 1 min under omrøring. Inkuber 2 timer ved 60 ° C. Skyl grundigt med DEPC-behandlet H2O Wrap instrumentet i aluminium og sat ind i instrumentet kassen. Wrap kassen. Steriliseres ved Autoklave.
  10. Rengøring af elektroforeseapparat: Vask apparatet, bakken og 15-brønde kam. Inkuber 15 min i 3% brintoverilte. Skyl gang med 100% ethanol. Lufttørre.
  11. Rengøring af glas retter: Rens glasfade RBS 2% hele natten, og derefter skylles med destilleret vand, før sterilisering ved 200 ° C.

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Representative Results

Proceduren Journal (figur 1) giver os inddrive eksemplarer af nethinden fra den kirurgiske blok til oprenset RNA og analysere transkriptomet af nethindeløsning. Nethindeløsning resulterer i opregulering af monocytkemotaktisk MCP1 gen CCL2, og faldet i ekspressionen af stangen fotoreceptor transducerende gen GNAT1, kortbølge kegle opsin OPN1SW og homeogene CRX (figur 2). Induktionen af CCL2 er resultatet af betændelse 9, mens den ledsagende nedsættelse af GNAT1, OPN1SW og CRX er resultatet af fotoreceptordegenerering begge stave og tappe. Tabet af kegler kan skyldes tab af ekspression af NXNL1, som koder for en stang-afledt Cone Levedygtighed Factor 7,10, eller dets paralog RdCVF2, som kodes af NXNL2 genet. Overraskende NXNL2 messenger exister i to forskellige versioner. Version 1 (NM_001161625.1) er en kodende sekvens afledt af phylogenic analyse, men er ikke tidligere blevet rapporteret at blive udtrykt 11, mens version 2 (NM_145283.2), hvor flere EST'er er blevet identificeret, er en unormal mRNA, udelukker den anden exon af genet og indeholder et alternativ andet exon, som indeholder en repetitiv Alu-sekvens, der ligger mere end 40 kb i 3'-retningen (fig. 3a). Anvendelse af RNA oprenset fra human nethinde, kan vi nu rapporteret, at de to versioner af NXNL2 mRNA udtrykt (figur 3b).

Figur 1
Figur 1. Billede af papkassen indeholder materialet fra Journal.

Figur 2
Figur 2.. Repræsentation af ekspressionen af en delmængde af gener ved hjælp Retinobase. For de gener der vises i disse radar grafer, CCL2, GNAT1, OPN1SW og CRX, den højre del af figuren svarer til RNA fra prøver af retinale afdelinger (RD1-18), mens den venstre del (NR1-18), er RNA fra alder matchede kontroller tilberedt med post mortem nethinder. Radaren graf metode er beskrevet i 8..

Figur 3
Figur 3. Ekspression af de to versioner af den NXNL2 genet i nethinden. en. Skematisk repræsentation af NXNL2 genet på kromosom 9.. NXNL2v1 har to exoner, der er forudsagt af multipel tilpasning og phylogenic analyse. NXNL2v2 mangler at andet exon og indbefatter en alternativ exon 2 ', der ligger> 40 kb i 3'-retningen. Than pile viser positionen af den anvendte primer. b.. RT-PCR viser ekspressionen af både NXNL2v1 og NXNL2v2 i nethinden. De rigtige baner svarer til reaktion i fravær af revers transkriptase. ACTB cytoplasmisk actin. Anvendte primere: NXNL2v1: 5'-GCATGAGCTGAGGAAGAGGT-3 ', 5'-CTCA AACGGAGAAATTCTGGA-3', NXNLv2: 5'-TCTGCACCCCCACGTTTATT-3 ', 5'-AGGGCCTCCT TTTCCATCTA-3'.

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Discussion

Udviklingen af ​​en procedure for væv opsving fra den kirurgiske blok har været afgørende for den transkriptomet analyse af nethindeløsning. Man bør bemærke, at denne type kirurgi praktiseres i en nødsituation, og at øjenlæger opererer har lidt tid til at deltage i et biologisk forskningsprogram, når de opererer. Denne retinectomy er også udført stokastisk i hver tjeneste, således at den nemmere måde at nå statistiske tal er at arbejde med et netværk. I dette netværk, er standardiseringen af ​​væv indsamlingen afgørende succes den biologiske analyse. Ved at give et materiale, meget let at bruge og præcise instrukser, som kan opbevares ved stuetemperatur i en operation kabinet, tæt på det kirurgiske blok, vi har tilskyndet kirurgerne til at deltage i vores undersøgelse.

Desuden blev standardisering af rensning af RNA opnåede at få det bedste af disse dyrebare clinical prøver. Samlinger af rene RNA'er kan opbevares i årevis ved -80 ° C og vil blive anvendt til yderligere undersøgelser som nye genomteknologier opstå. Protokollen er fastsat retinal kirurgisk prøve men kan også anvendes med held til at isolere RNA fra gnaver nethinde efter dissektion. Hvis RNA nedbrydes, a) kontrollere pH af CsCl / EDTA-opløsning. b) at gøre alle de løsninger fra ubrugte kemikalier. Den vigtigste begrænsning af teknikken er mængden af ​​udgangsmateriale, som bør svare til mindst 50.000 celler.

Hvad adskiller den anvendte metode her fra de fleste kommercielle reagenser leveret at isolere total RNA er graden af ​​renhed. RNA udtømt for enhver DNA-kontaminering, som eliminerer behovet for at bruge DNAse behandling, der kan være til skade for en yderligere procedure. Desuden er de samlede RNA præparater her forarmet i tRNA, der er kendt for at være kraftige inhibitorer af RNA-polymeraser, der almindeligvis anvendes iamplifikationstrinnet før hybridisering til microarray chips. Vi har observeret, at de prober syntetiseret ud fra disse RNA-præparater har en meget høj specifik aktivitet. Renhedsgraden af ​​RNA fremstillet ifølge metoden beskrevet her er meget velegnet til microarray hybridisering, men også til at konstruere cDNA biblioteker af høj kvalitet og for RNA-sekventering, som vi har observeret. Laboratoriet skal være RNAse gratis. PH af CsCl / EDTA bør være sure at undgå nedbrydning af RNA ved alkalisk lysering. Tætheden af ​​CsCl / EDTA bør omhyggeligt kontrolleres for ikke at være for høj, og for at forhindre sedimentering af RNA.

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Disclosures

Forfatterne har ikke noget at afsløre.

Acknowledgments

Vi takker Sacha Reichman og Dominique Santiard-Baron for deres hjælp med at redigere RNA oprensningsprotokol.

Materials

Name Company Catalog Number Comments
Centrifuge Beckman Coulter Aventi J-E
Rotor Beckman Coulter JS-5.3
Ultracentrifuge Beckman Coulter LE 80K
Rotor Beckman Coulter SW41
Power supply Biorad Pac 3000
PolytronTM Kinematica PT 2100 Supplied with PT-DA 2105:2
Agarose gel electrophoresis device Biorad MiniGel Cell GT
Imaging System Biorad GelDoc-It Imager
5 ml sterile polyethylene tube Greiner 115261
Sterile polyallomer centrifuge tube Beckman Coulter 331372
Chemical reagents Sigma Molecular biology grade (RNase and DNase free products)
Cleaning Solution VWR RBS
Microarray chip Affymetrix Human U133 plus 2 array

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References

  1. Mitry, D., Charteris, D. G., et al. The epidemiology of rhegmatogenous retinal detachment: geographical variation and clinical associations. The British Journal of Ophthalmology. 94 (6), 678 (2010).
  2. Green, R. E., Krause, J., et al. A draft sequence of the Neandertal genome. Science. 328 (5979), 710 (2010).
  3. Glisin, V., Crkvenjakov, R., et al. Ribonucleic acid isolated by cesium chloride centrifugation. Biochemistry. 13 (12), 2633 (1974).
  4. Van Gelder, R. N., von Zastrow, M. E., et al. Amplified RNA synthesized from limited quantities of heterogeneous cDNA. Proceedings of the National Academy of Sciences of the United States of America. 87 (5), 1663 (1990).
  5. Cavalieri, L. F., Yamaura, I. E. coli tRNAs as inhibitors of viral reverse transcription in vitro. Nucleic Acids Research. 2 (12), 2315 (1975).
  6. Sawadogo, M. On the inhibition of yeast RNA polymerases A and B by tRNA and alpha-amanitin. Biochemical and biophysical research communications. 98 (1), 261 (1981).
  7. Delyfer, M. N., Raffelsberger, W., et al. Transcriptomic analysis of human retinal detachment reveals both inflammatory response and photoreceptor death. PloS ONE. 6 (12), e28791 (2011).
  8. Kalathur, R. K., Gagniere, N., et al. RETINOBASE: a web database, data mining and analysis platform for gene expression data on retina. BMC Genomics. 9, 208 (2008).
  9. Nakazawa, T., Hisatomi, T., et al. Monocyte chemoattractant protein 1 mediates retinal detachment-induced photoreceptor apoptosis. Proceedings of the National Academy of Sciences of the United States of America. 104 (7), 2425 (2007).
  10. Leveillard, T., Sahel, J. A. Rod-derived cone viability factor for treating blinding diseases: from clinic to redox signaling. Science Translational Medicine. 2 (26), 26ps16 (2010).
  11. Chalmel, F., Leveillard, T., et al. Rod-derived Cone Viability Factor-2 is a novel bifunctional-thioredoxin-like protein with therapeutic potential. BMC Molecular Biology. 8, 74 (2007).

Tags

Genetik medicin molekylærbiologi Biomedical Engineering anatomi fysiologi neurobiologi Neuroscience Biochemistry Genomics Transcriptomics Ophthalmology Øjensygdomme neurovidenskab retina menneskelige vævsprøver RNA oprensning microarray analyse microarray Journal RNA DNA oprensning gelelektroforese sekventering kliniske applikationer kliniske teknikker
Transkriptom analyse af menneskets retinale kirurgiske prøver vha. Journal
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Delyfer, M. N., Aït-Ali, N.,… More

Delyfer, M. N., Aït-Ali, N., Camara, H., Clérin, E., Korobelnik, J. F., Sahel, J. A., Léveillard, T. Transcriptomic Analysis of Human Retinal Surgical Specimens Using jouRNAl. J. Vis. Exp. (78), e50375, doi:10.3791/50375 (2013).

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