Gli autori desiderano ringraziare Michael Alifrangis e Ulla Abildtrup per la genotipizzazione di parassiti e Christina Holm per un&#39;eccellente assistenza tecnica. Questo lavoro è stato finanziato da Howard Hughes Medical Institute (sovvenzione 55.005.511), la Fondazione Lundbeck (sovvenzione R9-A840) e dalla Fondazione Niels Bohr.

1. Generazione di appena selezionati eritrociti infetti Per questo test, i risultati migliori si ottengono utilizzando colture appena selezionati per espressione di superficie di PfEMP1 proteine. In questo esperimento particolare, il P. 3D7 lineage falciparum è stato selezionato utilizzando anticorpi specifici come descritto in precedenza 1. Giorno 1 <ol><li> Harvest 200 pl di globuli confezionati da un P. falciparum coltura contenente 2-5% IE fase tardiva mediante centrifugazione a 800 xg per 8 minuti a temperatura ambiente. </li><li> Risospendere il pellet sangue in 2 ml di 37 ° C caldo soluzione 0,75% di gelatina in una provetta sterile 14 ml, e lasciare per 15-20 minuti a 37 ° C per consentire l&#39;IE non infette e anello fase sedimenti. </li><li> Raccogliere il ritardo-fase IE, cioè la fase superiore, in una nuova provetta 14 ml e lavare due volte con 10 ml di 37 ° C caldi RBC-lavaggio media. </li><li> Diluire 50 ml di anticorpi specifici anti-PfEMP1 anticorpi in 2 ml di 37 ° C caldo RBC-lavaggio mezzi e filtro sterili. </li><li> Mescolare il lavato all&#39;ultimo stadio IE con gli antisieri sterilizzato diluito in un tubo 14 ml sterile e incubare per 30 min a 37 ° C con agitazione. </li><li> Centrifugare a 800 xg per 8 minuti a temperatura ambiente, e lavate due volte con 10 ml di 37 ° C caldi RBC-lavaggio media. </li><li> Lavare 50 pl di proteina Dynabead Una sospensione due volte con RBC-lavaggio supporti tramite un DynaMaq-15 magnete. </li><li> Risospendere le Dynabeads in 300 ml RBC-lavaggio dei media e aggiungerli alla lavato fase avanzata di IE. Incubare per 30 min a 37 ° C con agitazione. </li><li> Trasferire il tubo al DynaMaq-15 magnete. Lasciare agire per 1-2 minuti e rimuovere tutto il liquido. </li><li> Risospendere le Dynabeads in 4-5 ml di RBC-lavaggio dei media. Trasferire il tubo sul magnete e lasciare per 1-2 minuti prima di rimuovere tutto il liquido. Ripetere la fase di lavaggio una volta. </li><li> Trasferire le perle lavate a un cm 25 2 f cultura sterileLASK contenente 200 ul cellule rosse del sangue appena confezionati. Aggiungere 5-6 ml di Albumax supporto nella cultura. Memorizzare una notte a 5% di CO 2 a 37 ° C. </li></ol> Giorno 2 <ol start="12"><li> Rimuovere i Dynabeads dalla cultura in cui il IE selezionato è reinvaded le cellule rosse del sangue fresco, trasferendo tutta la cultura in una provetta 14 ml sterile. Inserire il tubo sul DynaMaq-15 magnete e lasciar riposare per 1-2 minuti. Poi, versare tutto il liquido nuovamente ad un pallone di coltura. </li><li> Cultura per il minor numero possibile di cicli fino ad un parassitemia del 5-10% e parassiti sono al ringstage. </li><li> IE devono essere analizzate mediante FACS per assicurarsi che il fenotipo corretta superficie, cioè l&#39;espressione della proteina di entrambi and/orPF11_0008 PFD1235w è stato ottenuto 1. </li></ol> 2. Preparazione di sonde Le sonde di RNA antisenso sono stati generati dalle regioni più variabili della PFD1235w e la var PF11_0008 &lt;/ Em&gt; geni di P. falciparum 3D7 DNA genomico. DNA è stato amplificato mediante PCR e clonato nel vettore pSPT18 o 19 per la trascrizione, rispettivamente, secondo la descrizione del produttore (Roche). Le sonde (580 paia di basi (bp) e 590 bp di lunghezza) sono state marcate con digossigenina (DIG) o biotina mediante un kit DIG RNA etichettatura o biotina RNA Mix etichettatura, rispettivamente. Abbiamo confermato la specificità delle sonde sia mediante analisi Northern blot e da etichetta singola analisi FISH 1. 3. Striscio sottile e la fissazione di parassiti Prima di ibridazione in situ Giorno 1 Tutte le fasi vengono eseguite in un ambiente privo di RNasi e tutti i reagenti sono RNase-free o pre-trattato con dietil pirocarbonato (DEPC) o RNasi Zap (Invitrogen). Le diapositive e le marze di copertura devono essere puliti con alcool per rimuovere grasso residuo di produzione. È importante eseguire il protocollo senza pausa tra fasial fine di minimizzare il rischio di contaminazione nucleasi. <ol><li> Fare un film sottile di sangue con il metodo standard diffusione 3. Utilizzando la lente obiettivo 100x olio del microscopio, contare il IE e calcolare la percentuale di IE nella cultura. Verificare che le fasi parassiti sono in sincronia approssimativi, sul ring e prime fasi del ciclo di trofozoite IE. Questi sono i pre-replicazione, uni-nucleate fasi, esprimendo mRNA gene var e adatto per le prove singole di trascrizione FISH delle cellule di questo tipo. </li><li> Trasferire 50 microlitri della cultura parassita, ad una parassitemia del 5-10%, 1,5 ml di una RNase-free provetta Eppendorf. Centrifugare per 1 min a 2000 rpm a temperatura ambiente. </li><li> Rimuovere il supporto e aggiungere 50 microlitri di PBS pH 7.2 in DEPC-acqua trattata. Agitare delicatamente il tubo. Ripetere il passaggio centrifuga sedimentazione due volte per lavare le cellule prive di mezzi di comunicazione e detriti cellulari. </li><li> Fai quattro buona qualità strisci sottili. Per ogni striscio, fare uno striscio sul&#39;altro di 11 mm di diametro e di una slitta 4 bene, vale a dire quattro lastre di vetro in totale, in una RNasi-free cofano (un passaggio fondamentale). Onda scivola brevemente in aria per asciugare. Effettuare tre diapositive supplementare per diapositiva controlli negativi-one per la colorazione singola sonda per qualsiasi altro gene irrilevante utilizzando una sonda specifica, un&#39;altra diapositiva per il trattamento RNasi e una terza slitta contenente IE non esprime il gene di interesse. Lasciare i vetrini si asciughino in panchina per 10-20 min. </li><li> Fissare i film sottili aggiungendo 60 pl di soluzione di fissazione contenente paraformaldeide al 4% e il 5% di acido acetico glaciale nei pozzetti. Lasciare agire per 10 minuti in panchina a temperatura ambiente. </li><li> Lavare i vetrini in 2x SSPE tampone per 5 minuti agitando con prudenza a temperatura ambiente. Brevemente rimuovere il liquido in eccesso premendo i pozzetti contenenti le cellule delicatamente con un fazzoletto di carta. </li><li> Coprire i vetrini con il 0,01% di pepsina in HCl 0,01 M per 2 minuti a 37 ° C (un passo estremamente critico). Lavare i vetrini in 2x SSPE per 5 min a temperatura ambiente. </li><li> Diluire la soluzione RNasi ad una concentrazione finale di 10 pg / ml. Coprire gli strisci di controllo negativo con il 60 microlitri della soluzione del RNasi. Incubare per 30 min a 37 ° C e poi lavare i vetrini in 2x SSPE per 5 min a temperatura ambiente. </li><li> Procedere immediatamente alla fase di ibridazione. </li></ol> 4. Ibridazione di sonde di RNA a diapositive fisse Giorno 1 <ol><li> Preparare la camera di ibridazione, come ad esempio un Thermostar HC4 100 o una scatola vuota puntale messo in un forno pulito, bagnandoli con RNasi Zap e asciugandosi pulirlo con un fazzoletto di carta. Riempire i canali della camera di ibridazione o il fondo della scatola con acqua trattata con DEPC per assicurarsi che i vetrini non si asciughino durante l&#39;ibridazione. Chiudere la camera e preriscaldare a 48 ° C. </li><li> Diluire le sonde nella soluzione di ibridazione ad una concentrazione finale di 12 ng / pl. Denaturare la soluzione della sonda a 65 ° C per 5 minuti in un blocco di riscaldamento. Immediatamente raffreddare su ghiaccio. </li><li> Brevemente l&#39;aria asciugare i vetrini dopo il lavaggio 2x SSPE. Rimuovere con cura il liquido in eccesso intorno ai pozzetti con un fazzoletto di carta. </li><li> Aprire la camera di ibridazione e posizionare i vetrini nella camera. Applicare una o entrambe le sonde in un volume totale di 60 microlitri per pozzetto. Coprire attentamente la goccia liquido con un RNasi-free coprioggetto utilizzando pinze sterili. </li><li> Chiudere la camera e ibridare i vetrini a 48 ° C per almeno 16 ore durante la notte. </li></ol> 5. Coniugazione anticorpo e Amplificazione Fluorescence Giorno 2 Le seguenti operazioni sono basate sulla TSA Plus System Palette fluorescenza da Perkin Elmer. Tutti i passaggi sono eseguiti a temperatura ambiente. <ol><li> Lavare i vetrini 3 volte in TNT tampone per 5 minuti con agitazione. </li><li> Bloccare i pozzetti in 150 ml di tampone per 30 min TNB. </li><li> Diluire il coniugato HRP-α-DIG anticorpo in tampone TNB in ​​un rapporto di 1:100 e il coniugato HRP-α-biotina in tampone TNB, con un rapporto di 1:500. Rimuovere il tampone in eccesso TNB dai vetrini con un fazzoletto di carta. Quando si rilevano due trascritti simultaneamente, entrambi gli anticorpi può andare in una sola volta. Applicare una quantità totale di 100 microlitri della soluzione di anticorpo-TNB per bene e coprire accuratamente con un vetrino coprioggetti. Incubare i vetrini per 2 ore. </li><li> Rimuovere i vetrini con attenzione. Lavare i vetrini in TNT tampone 3 volte per 5 min. </li><li> Diluire il FITC-fluoroforo nel reagente di amplificazione tiramide in un rapporto di 1:500 e applicare 100 pl della soluzione ad ogni pozzetto. Coprire i pozzetti con i vetrini coprioggetto e incubare per 12 min. </li><li> Togliere il coperchio scivola con cura e lavare i vetrini 3 volte per 5 min in tampone TNT agitando con prudenza. </li><li> Diluire il Cyan3-fluoroforo nel reagente di amplificazione tiramide ad un rapporto di 1:500. Aggiungere 100 microlitri perbene di questa soluzione. Coprire i pozzetti con i vetrini coprioggetto e incubare per 12 min. </li><li> Togliere il coperchio scivola attentamente e poi sciacquare i vetrini rapidamente per immersione in tampone TNT. </li><li> Lavare i vetrini 3 volte con tampone di TNT per 5 min. </li><li> Immergere rapidamente i vetrini in acqua trattata con DEPC. </li><li> Lasciare i vetrini asciugare all&#39;aria. Montare i vetrini con anti-fade reagente contenente DAPI. Sigillare gli angoli dei vetrini coprioggetto con smalto. </li><li> I vetrini sono ora pronti per la microscopia. Conservare i vetrini coperti con un foglio di alluminio e conservare a 4 ° C se l&#39;esperimento deve essere completata il giorno successivo. Una sigillatura completa dei lati dei vetrini possono essere effettuati 24 ore dopo l&#39;applicazione del reagente anti-fade. Ciò permetterà le diapositive da acquisire fino ad una settimana dopo il protocollo è stato completato. </li></ol> 6. Visualizzazione dei prodotti ibridati <ol><li> Accendere il microscopio. Un microscopio standard di immunofluorescenza è sufficient per questo tipo di esperimento, ma se si ha accesso ad un microscopio confocale è possibile utilizzare anche questo per le immagini in 3D o per ottenere i video delle vostre cellule colorate. Lasciare microscopio per riscaldare per 15-30 min. Accendere il computer e il software. </li><li> Verificare la qualità della colorazione sul microscopio immunofluorescenza. Scegli un posto che contiene pochi parassiti. </li><li> Regolare il guadagno di ogni laser manualmente. Regolare il pixel sosta a 4-6 m 1 s-1 e le misure di 512 x 512 pixel. </li><li> Scattare una foto di prova con cornice Lambda. Regolare il guadagno laser. </li><li> Prendete un minimo di 20 belle foto di singole cellule fluorescenti. Almeno 2-5 immagini di un campo contenente 5-20 parassiti sono necessari al fine di ottenere un quadro delle percentuale di parassiti positivi. </li></ol>

<ol>
	<li>Joergensen, L., Bengtsson, D. C., Bengtsson, A., Ronander, E., Berger, S. S., Turner, L., Dalgaard, M. B., Cham, G. K., Victor, M. E., Lavstsen, T., Theander, T. G., Arnot, D. E., Jensen, A. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Surface+co-expression+of+two+different+PfEMP1+antigens+on+single+Plasmodium+falciparum-infected+erythrocytes+facilitates+binding+to+ICAM1+and+PECAM1.">Surface co-expression of two different PfEMP1 antigens on single Plasmodium falciparum-infected erythrocytes facilitates binding to ICAM1 and PECAM1.</a> PLoS Pathog. 2, 1001083 (2010).</li><li>Pinaud, R., Mello, C. V., Velho, T. A., Wynne, R. D., Tremere, L. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+two+mRNA+species+at+single-cell+resolution+by+double-fluorescence+in+situ+hybridization.">Detection of two mRNA species at single-cell resolution by double-fluorescence in situ hybridization.</a> Nat. Protoc. 3, 1370-1379 (2008).</li><li>Cheeseborough, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> District Laboratory Practice in Tropical Countries. , (2006).</li><li>Brolin, K. J., Ribacke, U., Nilsson, S., Ankarklev, J., Moll, K., Wahlgren, M., Chen, Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+transcription+of+duplicated+var2csa+gene+copies+in+individual+Plasmodium+falciparum+parasites.">Simultaneous transcription of duplicated var2csa gene copies in individual Plasmodium falciparum parasites.</a> Genome Biol. 10, R117 (2009).</li><li>Epp, C., Li, F., Howitt, C. A., Chookajorn, T., Deitsch, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+associated+sense+and+antisense+noncoding+RNAs+are+transcribed+from+the+var+gene+family+of+virulence+genes+of+the+malaria+parasite+Plasmodium+falciparum.">Chromatin associated sense and antisense noncoding RNAs are transcribed from the var gene family of virulence genes of the malaria parasite Plasmodium falciparum.</a> RNA. 15, 116-127 (2009).</li><li>Freitas-Junior, L. H., Hernandez-Rivas, R., Ralph, S. A., Montiel-Condado, D., Ruvalcaba-Salazar, O. K., Rojas-Meza, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Telomeric+heterochromatin+propagation+and+histone+acetylation+control+mutually+exclusive+expression+of+antigenic+variation+genes+in+malaria+parasites.">Telomeric heterochromatin propagation and histone acetylation control mutually exclusive expression of antigenic variation genes in malaria parasites.</a> Cell. 121, 25-36 (2005).</li><li>Obernosterer, G., Martinez, J., Alenius, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Locked+nucleic+acid-based+in+situ+detection+of+microRNAs+in+mouse+tissue+sections.">Locked nucleic acid-based in situ detection of microRNAs in mouse tissue sections.</a> Nature Protoc. 2, 1508-1514 (2007).</li><li>Li, F., Sonbuchner, L., Kyes, S. A., Epp, C., Deitsch, K. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nuclear+non-coding+RNA+are+transcribed+from+the+centromeres+of+Plasmodium+falciparum+and+are+associated+with+centromeric+chromatin.">Nuclear non-coding RNA are transcribed from the centromeres of Plasmodium falciparum and are associated with centromeric chromatin.</a> J. Biol. Chem. 283, 5692-5698 (2008).</li><li>Thompson, J., Sinden, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+Pbs21+mRNA+during+sexual+development+of+Plasmodium+berghei.">In situ detection of Pbs21 mRNA during sexual development of Plasmodium berghei.</a> Mol. Biochem. Parasitol. 68, 189-196 (1994).</li><li>Thompson, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+detection+of+RNA+in+blood-+and+mosquito-stage+malaria+parasites.">In situ detection of RNA in blood- and mosquito-stage malaria parasites.</a> Methods Mole Med. 72, 225-235 (2002).</li><li>Arnot, D. E., Ronander, E., Bengtsson, D. 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+progression+of+the+intra-erythrocytic+cell+cycle+of+Plasmodium+falciparum+and+the+role+of+the+centriolar+plaques+in+asynchronous+mitotic+division+during+schizogony.">The progression of the intra-erythrocytic cell cycle of Plasmodium falciparum and the role of the centriolar plaques in asynchronous mitotic division during schizogony.</a> Int. J. Parasitol. 41, 71-80 (2011).</li><li>Osborne, C. S., Chakalova, L., Brown, K. E., Carter, D., Horton, A., Debrand, E., Goyenechea, B., Mitchell, J. A., Lopes, S., Reik, W., Fraser, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Active+genes+dynamically+colocalize+to+shared+sites+of+ongoing+transcription.">Active genes dynamically colocalize to shared sites of ongoing transcription.</a> Nat. Genet. 36, 1065-1071 (2004).</li></ol>

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Analisi FISH RNA, a differenza di metodi come Northern blotting e RT-PCR, permette la discriminazione di mRNA trascritti specifici a livello di singola cellula. Ciò rende possibile discriminare tra cellule trascrizionalmente attiva e inattiva, in questo esempio, P. falciparum protozoi parassiti all&#39;interno globuli rossi umani. Tali cellule intere osservazioni sono spesso necessari e possono svelare modelli trascrizionali importanti e nuovi 1. Sebbene altri metodi FIS...

<table border="1"><tbody><tr><td> Nome del reagente </td><td> Azienda </td><td> Numero di catalogo </td><td> Commenti </td></tr><tr><td> Acido acetico </td><td> Sigma / Aldrich </td><td> 338826-100ml </td><td></td></tr><tr><td> Albumax media: RPMI 1640 soluzione Glutammina </td><td> Lonza </td><td> BE12-115F </td><td> 500 ml RPMI 1640 5 ml di soluzione di glutammina </td></tr><tr><td> Albumax media: Gentamicina solfato Albumax soluzione </td><td> Lonza </td><td> BE02-012E </td><td> 2,5 ml di gentamicina solfato 50 ml di soluzione Albumax </td></tr><tr><td> Albumax-soluzione: ipoxantina </td><td> Sigma-Aldrich </td><td> H9377 </td><td> 0,8 g ipoxantina </td></tr><tr><td> Albumax-soluzione: Albumax II </td><td> Ionvitrogen </td><td> 11021-037 </td><td> 200 g Albumax </td></tr><tr><td> Albumax-soluzione: RPMI 1640 </td><td> Lonza </td><td> BE12-115F </td><td> 4 litro RPMI 1640 Sciogliere con magnete al max. 50 ° C. Filtro sterilizzare e conservare a -20 ° C in aliquote. </td></tr><tr><td> Amberlite </td><td> Sigma </td><td> A5710-110G </td><td> Resina deionizzare formammide. </td></tr><tr><td> Anti-biotina capra pAb Coniugato perossidasi </td><td> Calbiochem </td><td> 203206 </td><td></td></tr><tr><td> Anti-Dig anticorpo </td><td> Novus Biologicals </td><td> NB100-41330 </td><td></td></tr><tr><td> Anti-fade reagente con DAPI </td><td> Invitrogen </td><td> P36931 </td><td> I mezzi di montaggio deve curare per 24 h prima di sigillare il vetrino completamente. </td></tr><tr><td> Biotina RNA Labeling Mix </td><td&gt; Roche </td><td> 11685597910 </td><td></td></tr><tr><td> Fotocamera digitale </td><td> Digitale Nikon vista DC-F11 </td><td></td><td></td></tr><tr><td> Coprioggetto </td><td> Menzel-Glaser </td><td> 631-1570 </td><td></td></tr><tr><td> cultura pallone 25 cm 2 di superficie Nunclon </td><td> Nunc </td><td> 156340 </td><td></td></tr><tr><td> DEPC </td><td> Fluka / Sigma </td><td> 32490-100ml </td><td> 1 ml DEPC in 1000 ml di acqua deinonized. Aggiungere una barra di agitazione e mescolare per 12 ore. Autoclave per 30 min. </td></tr><tr><td> Macchina fotografica digitale </td><td> Digitale Nikon vista DC-F11 </td><td></td><td></td></tr><tr><td> Dynabeads proteina A </td><td> Invitrogen </td><td> 10002D </td><td></td></tr><tr><td> DynaMaq-15 Magnete </td><td> Invitrogen </td><td> 123-01D </td><td></td></tr><tr><td> Formammide bioultra 99% </td><td> Fluka/ Sigma </td><td> 47671-1l-F </td><td> Formammide deionizzata: 5 g di resina scambiatrice di ioni per 100 ml di formammide. Mescolare 30 min. Filtrare su carta Whatman. </td></tr><tr><td> . Gelatina 0 soluzione al 75%: Gelatina </td><td> Sigma-Aldrich </td><td> G2500 </td><td> 3,75 g di gelatina in 500 ml di RPMI 1640. Riscaldare a 56 ° C per sciogliere. Filtro sterilizzare quando 56 ° C. Conservare a -20 ° C in aliquote. </td></tr><tr><td> . Gelatina 0 soluzione al 75%: RPMI 1640 </td><td> Lonza </td><td> BE12-115F </td><td> 3,75 g di gelatina in 500 ml di RPMI 1640. Riscaldare a 56 ° C per sciogliere. Filtro sterilizzare quando 56 ° C. Conservare a -20 ° C in aliquote. </td></tr><tr><td> Glutammina soluzione: L-glutamina </td><td> Sigma-Aldrich </td><td> G3126 </td><td> 14,6 g L glutammina in 500 ml di NaCl allo 0,9%. Sciogliere, filtro sterilize e conservare a -20 ° C in aliquote. </td></tr><tr><td> Glutammina soluzione: HCl </td><td> Sigma </td><td> H1758 </td><td> 14,6 g L glutammina in 500 ml di NaCl allo 0,9%. Sciogliere, filtro sterilizzare e conservare a -20 ° C in aliquote. </td></tr><tr><td> Ibridazione soluzione: Formamide Bio ultra 99% SSC 20x </td><td> Fluka / Sigma </td><td> 47671-1l-F </td><td> Totale 20 ml, mantenere congelato a -20 ° C in aliquote 10 ml di formammide deionizzata </td></tr><tr><td> Ibridazione soluzione: concentrato 50x Denhardt </td><td> Sigma </td><td> D2532 </td><td> 5ml 20xSSC </td></tr><tr><td> Ibridazione soluzione: reagente lievito tRNARoche blocco </td><td> Sigma </td><td> R-6750 </td><td> 2 ml 50x Denhardt 250 microlitri 20 mg al lievito tRNA ml </td></tr><tr><td><td> Fluka / Sigma </td><td> 31149-106GF </td><td> 0.4g Roche blocco reagente 1 ml di 10 mg per ml DNA di sperma di salmone (Critical: denaturano salmone spermDNA a 96 ° C per 5 minuti prima di aggiungere alla soluzione di ibridazione) </td></tr><tr><td> Hybridizer Thermostar 100 HC4 </td><td> Quantifoil Instruments GmbH </td><td> 1004-0011 </td><td> Può essere sostituito da forno di ibridazione e camere di ibridazione RNasi libero imbottiti con acqua DEPC. </td></tr><tr><td> Olio di immersione UV trasparente fluorescenza libero </td><td> Sigma </td><td> 10976-1EA </td><td></td></tr><tr><td> Microscopio confocale o Immunofluorescenza </td><td> Nikon D-Eclipse TE2000C </td><td></td><td></td></tr><tr><td> Nailpolish </td><td> Disponibile in farmacia </td><td></td><td></td></tr><tr><td> PBS 20x: NaCl KCl Na 2 HPO 4 x 2H 2 O KH 2 PO 4 DEPC deionizzata H 2 O </td><td></td><td></td><td> ASOLO pH a 7,4 160 g 4 g 23 g 4 g 1 l </td></tr><tr><td> Paraformaldeide 4% </td><td> Fluka / chemika </td><td> 76240 </td><td> 4 g PFA in 80 ml di PBS / DEPC. Riscaldare a 65 ° C fino alla PFA dissolve. Aggiungere 20 ml di PBS, lasciare che la soluzione si raffreddi. Regolare il pH a 7,4. Filtro. Conservare in aliquote a -20 ° C. </td></tr><tr><td> Paraformaldeide al 4% / acido acetico al 5% </td><td></td><td></td><td> 950 microlitri paraformaldhyde + 50 microlitri di acido acetico </td></tr><tr><td> Pepsina </td><td> Sigma / Aldrich </td><td> P7000 </td><td></td></tr><tr><td> Perossidasi-coniugato anti-biotina </td><td> Calbiochem </td><td> 203206 </td><td></td></tr><tr><td> RBC-wash media: RPMI 1640 soluzione Glutammina </td><td> Lonza </td><td> BE12-115F </td><td> 500 ml RPMI 1640 5 ml soluzione glutammina </td></tr><tr><td> RBC-wash media: Gentamicina solfato </td><td> Lonza </td><td> BE02-012E </td><td> 2,5 ml di gentamicina solfato </td></tr><tr><td> RNasi </td><td> Sigma / Aldrich </td><td></td><td> Fai un 10 mg / ml di soluzione concentrata. </td></tr><tr><td> RNasi libero 1,5 ml tubo </td><td> Ambion </td><td> AM12450 </td><td></td></tr><tr><td> RNasi Zap </td><td> Ambion </td><td> AM9780 </td><td></td></tr><tr><td> Slides 4 pozzetti 11 millimetri </td><td> Thermo Scientific </td><td> MENZXER306W </td><td></td></tr><tr><td> SSC 20x: NaCl </td><td> Sigma / AlAldrich </td><td> S9625 </td><td> 3 M NaCl (175 g / l) 0,3 M Na 3 citrato x H 2 O (88 g / l) Regolare il pH a 7,0 con HCl 1M </td></tr><tr><td> SSC 20x: citrato di sodio diidrato </td><td> Sigma / Aldrich </td><td> W302600 </td><td> 3 M NaCl (175 g / l) 0,3 M Na 3 citrato x H 2 O (88 g / l) Regolare il pH a 7,0 con HCl 1M </td></tr><tr><td> SSPE 20x: NaCl </td><td> Sigma / Aldrich </td><td> S9625 </td><td> 175,3 g NaCl </td></tr><tr><td> SSPE 20x: NaH 2 PO 4 </td><td> Sigma / Aldrich </td><td> S0751 </td><td> 27,6 g di NaH 2 PO 4. </td></tr><tr><td> SSPE 20x: 4 EDTA in polvere </td><td></td><td></td><td> 9,4 g di polvere EDTA Aggiungere acqua DEPC, correggere il pH 7.4. Autospaccò per 20 min </td></tr><tr><td> TNB buffer: buffer di TNT </td><td></td><td></td><td> 10 ml tampone TNT </td></tr><tr><td> TNB buffer: reagente di blocco </td><td></td><td></td><td> 0,05 g di reagente di blocco Per sciogliere il reagente bloccante, riscaldare la soluzione a 60 ° C per un&#39;ora sotto agitazione. Conservare a -20 ° C. (Derivato dalla Perkin Elmer TSA-protocollo.) </td></tr><tr><td> TNT tampone: Tris / HCl </td><td> Sigma </td><td> T1503 </td><td> 1M Tris / HCl, pH 8,0 </td></tr><tr><td> TNT buffer: NaCl </td><td> Sigma Aldrich </td><td> S9625 </td><td> 100 ml </td></tr><tr><td> TNT buffer: Tween20 </td><td> Sigma </td><td> 93773 </td><td> 5 M NaCl 30 ml 1 ml DEPC dH 2 O869 Ml Portare il pH a 7,5 a temperatura ambiente. (Derivato dalla Perkin Elmer TSA-protocollo.) </td></tr><tr><td> TSA più Cyanine3 / fluoresceina sistema </td><td> Perkin Elmer </td><td> NEL753000IKT </td><td> Leggi il protocollo dal TSA Plus System Palette fluorescenza con attenzione prima di iniziare l&#39;esperimento. </td></tr><tr><td> Tubi 14 ml sterile </td><td> Almeco - CM Aps LAB </td><td> 91016 </td><td></td></tr></tbody></table>

analysis of single-cell gene transcription by rna fluorescent in situ hybridization (fish)

Adesione di Plasmodium falciparum eritrociti infetti (IE) per recettori umani endoteliali durante la malaria è mediato dall&#39;espressione di PfEMP1 varianti proteiche codificate dai geni var. Il aploide P. falciparum genoma ospita circa 60 differenti geni var di cui uno solo è creduto per essere trascritto per cella alla volta durante la fase di sangue dell&#39;infezione. Come tale regolamentazione si escludono a vicenda della trascrizione genica var si ottiene non è chiaro, così come l&#39;identificazione di singoli geni var o sotto-gruppi di geni associati con var diversi recettori e la conseguenza del legame differenziale sui risultati clinici di P. falciparum infezioni. Di recente, il paradigma trascrizione si escludono a vicenda è stata messa in dubbio da saggi di trascrizione sulla base di singoli P. falciparum identificazione trascrizione in inf singoloected cellule eritrocitari utilizzando RNA ibridazione fluorescente in situ (FISH) Analisi della trascrizione genica var dal parassita in singoli nuclei di P. falciparum IE 1. Qui, presentiamo un protocollo dettagliato per effettuare la RNA-FISH metodologia di analisi della trascrizione genica var in single-nuclei di P. falciparum infettato eritrociti umani. Il metodo si basa sull&#39;uso di digossigenina e biotina marcato con sonde di RNA antisenso utilizzando il sistema TSA Plus. Fluorescence Palette 2 (Perkin Elmer), analisi al microscopio e appena selezionato P. falciparum IE. Il metodo in situ può essere utilizzato per monitorare la trascrizione e la regolazione di una varietà di geni espressi durante le diverse fasi del P. ciclo di vita falciparum ed è adattabile ad altre specie parassita della malaria e di altri organismi e tipi di cellule.

La figura 1 illustra un diagramma di flusso delle fasi principali e la durata dell&#39;impulso del metodo FISH RNA. Una serie di immagini rappresentative di bene e male esperimenti mRNA conservati colorate FISH utilizzando una singola P. falciparum IE sono mostrati in Figura 2. Questo protozoo intracellulare ha una piccola (1-1,5 micron di diametro), nucleo che è di colore blu con DAPI. Ventiquattro ore dopo l&#39;invasione degli eritrociti in ...

Watch this Scientific Journal Video about Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH at JoVE.com

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Fluorescente<em&gt; In situ</emIbridazione&gt; (FISH) per identificare trascritti di mRNA in cellule singole consente analisi dell&#39;attività poligenici come la trascrizione simultanea di più di un membro della<em&gt; Var</em&gt; Multigene famiglia in<em&gt; Plasmodium falciparum</em&gt; Eritrociti infetti<sup&gt; 1</sup&gt;. La tecnica è adattabile e può essere utilizzato su diversi tipi di geni, cellule e organismi.

Analisi di Single-cell trascrizione del gene per l&#39;RNA fluorescente<em&gt; In Situ</em&gt; Ibridazione (FISH)

Adhesion of Plasmodium falciparum infected erythrocytes (IE) to human endothelial receptors during malaria infections is mediated by expression of PfEMP1 ...

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JoVE Journal

Biology

Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization (FISH)

15.3K Views.  University of Copenhagen. Fluorescente In situ (FISH) per identificare trascritti di mRNA in cellule singole consente analisi dell'attività poligenici come la trascrizione simultanea di più di un membro della Var Multigene famiglia in Plasmodium falciparum Eritrociti infetti 1. La tecnica è adattabile e può essere utilizzato su diversi tipi di geni, cellule e organismi.

Video: Analysis of Single-cell Gene Transcription by RNA Fluorescent In Situ Hybridization FISH

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. The protocol is a modified version of the standard in situ hybridization using alkaline phosphatase and substrates such as NBT/BCIP and Fast Red 1,2. This protocol utilizes standard digoxygenin and fluorescein labeled probes along with tyramide signal amplification (TSA) 3. The commercially available TSA kits allow flexible experimental design as fluorescence emission from green to far-red can be used in combination with various nuclear stains, such as propidium iodide, or fluorescence immunohistochemistry for proteins. TSA produces a reactive fluorescent substrate that quickly covalently binds to moieties, typically tyrosine residues, in the immediate vicinity of the labeled antisense riboprobe. The resulting staining patterns are high resolution in that subcellular localization of the mRNA can be observed using laser scanning confocal microscopy 3,4. One can observe nascent transcripts at the chromosomal loci, distinguish nuclear and cytoplasmic staining and visualize other patterns such as cortical localization of mRNA. Studies in Drosophila indicate that roughly 70% of mRNAs exhibit specific patterns of subcellular localization that frequently correlate with the function of the encoded protein 5. When combined with computer-aided reconstruction of 3D confocal datasets, our protocol allows the detailed analysis of mRNA distribution with sub-cellular resolution in whole vertebrate embryos.

Zebrafish Whole Mount High-Resolution Double Fluorescent In Situ Hybridization

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology. Here, we present a high-resolution double fluorescent in situ hybridization protocol for analyzing the precise expression pattern of a single gene and for determining the overlap of the expression domains of two genes. We include a propidium iodide nuclear counter-stain to highlight tissue organization.

Zebrafish intero monte ad alta risoluzione doppia fluorescente In Situ Ibridazione

Whole mount in situ hybridization is one of the most widely used techniques in developmental biology.  Here, we present a high-resolution double ...

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Biologia

Fluorescence in situ hybridization (FISH) Protocol in Human Sperm

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic process in the parents. In human males, these errors can lead to the production of spermatozoa with numerical chromosome abnormalities which represent an increased risk of transmitting these anomalies to the offspring.
For this reason, the technique of fluorescence in situ hybridization (FISH) on sperm nuclei has become a protocol widely incorporated in the context of clinical diagnosis. This practice provides an estimate of the frequencies of numerical chromosome abnormalities in the gametes of the patients that seek for genetic reproductive advice.
To date, the chromosomes most frequently included in sperm FISH analysis are chromosomes X, Y, 13, 18 and 21.
This video-article describes, step by step, how to process and fix a human semen sample, how to decondense and denature the sperm chromatin, how to proceed to obtain sperm FISH preparations, and how to visualize the results at the microscope. Special remarks of the most relevant steps are given to achieve the best results.

Fluorescence in situ hybridization FISH Protocol in Human Sperm

This video-article describes, step by step, how to process a semen sample to achieve good-quality fluorescence in situ hybridization on human spermatozoa. Preparations obtained can be used for aneuploidy screening in the context of clinical diagnosis.

Fluorescenza In situ</emIbridazione> (FISH) Protocollo di sperma umano

Aneuploidies are the most frequent chromosomal abnormalities in humans. Most of these abnormalities result from meiotic errors during the gametogenic ...

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence (T7, T3, SP6) and the gene to be transcribed (Figure 1A). A typical transcription reaction consists of the template DNA, RNA polymerase, ribonucleotide triphosphates, RNase inhibitor and buffer containing Mg2+ ions.
Large amounts of high quality RNA are often required for a variety of applications. Use of in vitro transcription has been reported for RNA structure and function studies such as splicing1, RNAi experiments in mammalian cells2, antisense RNA amplification by the "Eberwine method"3, microarray analysis4 and for RNA vaccine studies5. The technique can also be used for producing radiolabeled and dye labeled probes6. Warren, et al. recently reported reprogramming of human cells by transfection with in vitro transcribed capped RNA7. The T7 High Yield RNA Synthesis Kit from New England Biolabs has been designed to synthesize up to 180 &mu;g RNA per 20 &mu;l reaction. RNA of length up to 10kb has been successfully transcribed using this kit. Linearized plasmid DNA, PCR products and synthetic DNA oligonucleotides can be used as templates for transcription as long as they have the T7 promoter sequence upstream of the gene to be transcribed.
Addition of a 5' end cap structure to the RNA is an important process in eukaryotes. It is essential for RNA stability8, efficient translation9, nuclear transport10 and splicing11. The process involves addition of a 7-methylguanosine cap at the 5' triphosphate end of the RNA. RNA capping can be carried out post-transcriptionally using capping enzymes or co-transcriptionally using cap analogs. In the enzymatic method, the mRNA is capped using the Vaccinia virus capping enzyme12,13. The enzyme adds on a 7-methylguanosine cap at the 5' end of the RNA using GTP and S-adenosyl methionine as donors (cap 0 structure). Both methods yield functionally active capped RNA suitable for transfection or other applications14 such as generating viral genomic RNA for reverse-genetic systems15 and crystallographic studies of cap binding proteins such as eIF4E16.
In the method described below, the T7 High Yield RNA Synthesis Kit from NEB is used to synthesize capped and uncapped RNA transcripts of Gaussia luciferase (GLuc) and Cypridina luciferase (CLuc). A portion of the uncapped GLuc RNA is capped using the Vaccinia Capping System (NEB). A linearized plasmid containing the GLuc or CLuc gene and T7 promoter is used as the template DNA. The transcribed RNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity. Capped CLuc RNA is used as the internal control to normalize GLuc expression.

In vitro Transcription and Capping of Gaussia Luciferase mRNA Followed by HeLa Cell Transfection

This method describes high yield in vitro synthesis of both capped and uncapped mRNA from a linearized plasmid containing the Gaussia luciferase (GLuc) gene. The RNA is purified and a fraction of the uncapped RNA is enzymatically capped using the Vaccinia virus capping enzyme. In the final step, the mRNA is transfected into HeLa cells and cell culture supernatants are assayed for luciferase activity.

Trascrizione in vitro e tappatura mRNA Luciferase Gaussia Seguito da cellule HeLa Transfection

In vitro transcription is the synthesis of RNA transcripts by RNA polymerase from a linear DNA template containing the corresponding promoter sequence ...

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. Each of these features can be accomplished with in situ hybridization to mRNAs within cells. Here we present a radioactive in situ hybridization method modified from Clayton et al. (1988)1 that has been working successfully in our lab for many years, especially for adult vertebrate brains2-5. The long complementary RNA (cRNA) probes to the target sequence allows for detection of low abundance transcripts6,7. Incorporation of radioactive nucleotides into the cRNA probes allows for further detection sensitivity of low abundance transcripts and quantitative analyses, either by light sensitive x-ray film or emulsion coated over the tissue. These detection methods provide a long-term record of target gene expression. Compared with non-radioactive probe methods, such as DIG-labeling, the radioactive probe hybridization method does not require multiple amplification steps using HRP-antibodies and/or TSA kit to detect low abundance transcripts. Therefore, this method provides a linear relation between signal intensity and targeted mRNA amounts for quantitative analysis. It allows processing 100-200 slides simultaneously. It works well for different developmental stages of embryos. Most developmental studies of gene expression use whole embryos and non-radioactive approaches8,9, in part because embryonic tissue is more fragile than adult tissue, with less cohesion between cells, making it difficult to see boundaries between cell populations with tissue sections. In contrast, our radioactive approach, due to the larger range of sensitivity, is able to obtain higher contrast in resolution of gene expression between tissue regions, making it easier to see boundaries between populations. Using this method, researchers could reveal the possible significance of a newly identified gene, and further predict the function of the gene of interest.

Radioactive in situ Hybridization for Detecting Diverse Gene Expression Patterns in Tissue

This protocol is successfully used to quantitatively detect levels and spatial patterns of mRNA expression in multiple tissue types across vertebrate species. The method can detect low abundance transcripts and allows processing of hundreds of slides simultaneously. We present this protocol using expression profiling of avian embryonic brain formation as an example.

Radioattivo In situ Ibridazione per il rilevamento Diversi schemi di espressione genica nei tessuti

Knowing the timing, level, cellular localization, and cell type that a gene is expressed in contributes to our understanding of the function of the gene. ...

Detection of Viral RNA by Fluorescence in situ Hybridization (FISH)

Viruses that infect cells elicit specific changes to normal cell functions which serve to divert energy and resources for viral replication. Many aspects of host cell function are commandeered by viruses, usually by the expression of viral gene products that recruit host cell proteins and machineries. Moreover, viruses engineer specific membrane organelles or tag on to mobile vesicles and motor proteins to target regions of the cell (during de novo infection, viruses co-opt molecular motor proteins to target the nucleus; later, during virus assembly, they will hijack cellular machineries that will help in the assembly of viruses). Less is understood on how viruses, in particular those with RNA genomes, coordinate the intracellular trafficking of both protein and RNA components and how they achieve assembly of infectious particles at specific loci in the cell. The study of RNA localization began in earlier work. Developing lower eukaryotic embryos and neuronal cells provided important biological information, and also underscored the importance of RNA localization in the programming of gene expression cascades. The study in other organisms and cell systems has yielded similar important information. Viruses are obligate parasites and must utilise their host cells to replicate. Thus, it is critical to understand how RNA viruses direct their RNA genomes from the nucleus, through the nuclear pore, through the cytoplasm and on to one of its final destinations, into progeny virus particles 1.
FISH serves as a useful tool to identify changes in steady-state localization of viral RNA. When combined with immunofluorescence (IF) analysis 22, FISH/IF co-analyses will provide information on the co-localization of proteins with the viral RNA3. This analysis therefore provides a good starting point to test for RNA-protein interactions by other biochemical or biophysical tests 4,5, since co-localization by itself is not enough evidence to be certain of an interaction. In studying viral RNA localization using a method like this, abundant information has been gained on both viral and cellular RNA trafficking events 6. For instance, HIV-1 produces RNA in the nucleus of infected cells but the RNA is only translated in the cytoplasm. When one key viral protein is missing (Rev) 7, FISH of the viral RNA has revealed that the block to viral replication is due to the retention of the HIV-1 genomic RNA in the nucleus 8. 
Here, we present the method for visual analysis of viral genomic RNA in situ. The method makes use of a labelled RNA probe. This probe is designed to be complementary to the viral genomic RNA. During the in vitro synthesis of the antisense RNA probe, the ribonucleotide that is modified with digoxigenin (DIG) is included in an in vitro transcription reaction. Once the probe has hybridized to the target mRNA in cells, subsequent antibody labelling steps (Figure 1) will reveal the localization of the mRNA as well as proteins of interest when performing FISH/IF.

Detection of Viral RNA by Fluorescence in situ Hybridization FISH

A fluorescence in situ hybridization (FISH) method was developed to visually detect viral genomic RNA using fluorescence microscopy. A probe is made with specificity to the viral RNA that can then be identified using a combination of hybridization and immunofluorescence techniques. This technique offers the advantage of identifying the localization of the viral RNA or DNA at steady-state, providing information on the control of intracellular virus trafficking events.

Rilevamento di RNA virale per fluorescenza<em&gt; In situ</em&gt; Hybridization (FISH)

Viruses  that infect cells elicit specific changes to normal cell functions which serve  to divert energy and resources for viral replication. Many ...

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The specification of replication timing is thought to be a dynamic process regulated by tissue-specific and developmental cues that are responsive to epigenetic modifications. However, the mechanisms regulating where and when DNA replication initiates along chromosomes remains poorly understood. Homologous chromosomes usually replicate synchronously, however there are notable exceptions to this rule. For example, in female mammalian cells one of the two X chromosomes becomes late replicating through a process known as X inactivation1. Along with this delay in replication timing, estimated to be 2-3 hr, the majority of genes become transcriptionally silenced on one X chromosome. In addition, a discrete cis-acting locus, known as the X inactivation center, regulates this X inactivation process, including the induction of delayed replication timing on the entire inactive X chromosome. In addition, certain chromosome rearrangements found in cancer cells and in cells exposed to ionizing radiation display a significant delay in replication timing of &gt;3 hours that affects the entire chromosome2,3. Recent work from our lab indicates that disruption of discrete cis-acting autosomal loci result in an extremely late replicating phenotype that affects the entire chromosome4. Additional 'chromosome engineering' studies indicate that certain chromosome rearrangements affecting many different chromosomes result in this abnormal replication-timing phenotype, suggesting that all mammalian chromosomes contain discrete cis-acting loci that control proper replication timing of individual chromosomes5.
Here, we present a method for the quantitative analysis of chromosome replication timing combined with fluorescent in situ hybridization. This method allows for a direct comparison of replication timing between homologous chromosomes within the same cell, and was adapted from6. In addition, this method allows for the unambiguous identification of chromosomal rearrangements that correlate with changes in replication timing that affect the entire chromosome. This method has advantages over recently developed high throughput micro-array or sequencing protocols that cannot distinguish between homologous alleles present on rearranged and un-rearranged chromosomes. In addition, because the method described here evaluates single cells, it can detect changes in chromosome replication timing on chromosomal rearrangements that are present in only a fraction of the cells in a population.

Chromosome Replicating Timing Combined with Fluorescent In situ Hybridization

A quantitative method for the analysis of chromosome replication timing is described. The method utilizes BrdU incorporation in combination with fluorescent in situ hybridization (FISH) to assess replication timing of mammalian chromosomes. This technique allows for the direct comparison of rearranged and un-rearranged chromosomes within the same cell.

Cromosoma Timing Replica combinazione con fluorescente<em&gt; In situ</em&gt; Ibridazione

Mammalian DNA replication initiates at multiple sites along chromosomes at different times during S phase, following a temporal replication program. The ...

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Assessing the expression pattern of a gene, as well as the subcellular localization properties of its transcribed RNA, are key features for understanding its biological function during development. RNA in situ hybridization (RNA-ISH) is a powerful method used for visualizing RNA distribution properties, be it at the organismal, cellular or subcellular levels 1. RNA-ISH is based on the hybridization of a labeled nucleic acid probe (e.g. antisense RNA, oligonucleotides) complementary to the sequence of an mRNA or a non-coding RNA target of interest 2. As the procedure requires primary sequence information alone to generate sequence-specific probes, it can be universally applied to a broad range of organisms and tissue specimens 3. Indeed, a number of large-scale ISH studies have been implemented to document gene expression and RNA localization dynamics in various model organisms, which has led to the establishment of important community resources 4-11. While a variety of probe labeling and detection strategies have been developed over the years, the combined usage of fluorescently-labeled detection reagents and enzymatic signal amplification steps offer significant enhancements in the sensitivity and resolution of the procedure 12. Here, we describe an optimized fluorescent in situ hybridization method (FISH) employing tyramide signal amplification (TSA) to visualize RNA expression and localization dynamics in staged Drosophila embryos. The procedure is carried out in 96-well PCR plate format, which greatly facilitates the simultaneous processing of large numbers of samples.

Whole Mount RNA Fluorescent in situ Hybridization of Drosophila Embryos

Here we describe a whole-mount fluorescent in situ hybridization (FISH) protocol for determining the expression and localization properties of RNAs expressed during embryogenesis in the fruit fly, Drosophila melanogaster.

Tutta la Monte RNA fluorescente<em&gt; In situ</em&gt; L&#39;ibridazione di<em&gt; Drosophila</em&gt; Embrioni

Assessing  the expression pattern of a gene, as well as the subcellular localization  properties of its transcribed RNA, are key features for ...

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

Marine elasmobranchs are valued animal models for biomedical and genomic studies as they are the most primitive vertebrates to have adaptive immunity and have unique mechanisms for osmoregulation 1-3. As the most primitive living jawed-vertebrates with paired appendages, elasmobranchs are an evolutionarily important model, especially for studies in evolution and development. Marine elasmobranchs have also been used to study aquatic toxicology and stress physiology in relationship to climate change 4. Thus, development and adaptation of methodologies is needed to facilitate and expand the use of these primitive vertebrates to multiple biological disciplines. Here I present the successful adaptation of RNA whole mount in situ hybridization and histological techniques to study gene expression and cell histology in elasmobranchs.
Monitoring gene expression is a hallmark tool of developmental biologists, and is widely used to investigate developmental processes 5. RNA whole mount in situ hybridization allows for the visualization and localization of specific gene transcripts in tissues of the developing embryo. The expression pattern of a gene's message can provide insight into what developmental processes and cell fate decisions a gene may control. By comparing the expression pattern of a gene at different developmental stages, insight can be gained into how the role of a gene changes during development.
While whole mount in situ's provides a means to localize gene expression to tissue, histological techniques allow for the identification of differentiated cell types and tissues. Histological stains have varied functions. General stains are used to highlight cell morphology, for example hematoxylin and eosin for general staining of nuclei and cytoplasm, respectively. Other stains can highlight specific cell types. For example, the alcian blue stain reported in this paper is a widely used cationic stain to identify mucosaccharides. Staining of the digestive tract with alcian blue can identify the distribution of goblet cells that produce mucosaccharides. Variations in mucosaccharide constituents on short peptides distinguish goblet cells by function within the digestive tract 6. By using RNA whole mount in situ's and histochemical methods concurrently, cell fate decisions can be linked to gene-specific expression.
Although RNA in situ's and histochemistry are widely used by researchers, their adaptation and use in marine elasmobranchs have met limited and varied success. Here I present protocols developed for elasmobranchs and used on a regular basis in my laboratory. Although further modification of the RNA in situ's hybridization method may be needed to adapt to different species, the protocols described here provide a strong starting point for researchers wanting to adapt the use of marine elasmobranchs to their scientific inquiries.

RNA In situ Hybridization in Whole Mount Embryos and Cell Histology Adapted for Marine Elasmobranchs

By combining methods for RNA whole mount in situ hybridization and histology, gene expression can be linked with cell fate decisions in the developing embryo. These methods have been adapted to marine elasmobranchs and facilitate the use of these animals as model organisms for biomedical, toxicology and comparative studies.

RNA<em&gt; In situ</emIbridazione&gt; in embrioni Mount interi e istologia Adattato per Elasmobranchi Marine

Marine  elasmobranchs are valued animal models for biomedical and genomic studies as  they are the most primitive vertebrates to have adaptive immunity ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

Visualizzazione e l&#39;analisi di molecole di mRNA mediante fluorescenza<em&gt; In Situ</em&gt; Ibridazione in<em&gt; Saccharomyces cerevisiae</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Analysis of Global RNA Synthesis at the Single Cell Level following Hypoxia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a particular transcriptional program in order to mount an appropriate and coordinated cellular response. The cell possesses several oxygen sensor enzymes that require molecular oxygen as cofactor for their activity. These range from prolyl-hydroxylases to histone demethylases. The majority of studies analyzing cellular responses to hypoxia are based on cellular populations and average studies, and as such single cell analysis of hypoxic cells are seldom performed. Here we describe a method of analysis of global RNA synthesis at the single cell level in hypoxia by using Click-iT RNA imaging kits in an oxygen controlled workstation, followed by microscopy analysis and quantification.&nbsp; Using cancer cells exposed to hypoxia for different lengths of time, RNA is labeled and measured in each cell. This analysis allows the visualization of temporal and cell-to-cell changes in global RNA synthesis following hypoxic stress.

We describe a technique for analysis of global RNA synthesis in hypoxia using imaging. Click-chemistry labeling of RNA has not previously been performed under hypoxia and allows visualization of global RNA changes at the single cell level. This approach complements the existing averaged RNA techniques, allowing direct visualization of cell-to-cell changes in global RNA synthesis.

Analisi globale di RNA Sintesi a livello di singola cellula seguente ipossia

Hypoxia or lowering of the oxygen availability is involved in many physiological and pathological processes. At the molecular level, cells initiate a ...