Gli autori ringraziano la signora Jennifer M. Lommel e Tara N. Rognan per la manutenzione degli impianti di Arabidopsis. Questo lavoro è stato sostenuto da fondi di start-up da Saint Louis University e il National Institutes di borse di Salute 1R15GM086846-01 e 3R15GM086846-01S1 a W. Xiao.

I. genetica Crossing 1. Castrante il genitore femmina Al fine di distinguere gli alleli materni e paterni, utilizzando sequenza polimorfismo del DNA, due ecotipi diversi, ad esempio, la Columbia-0 (Col-0) e Ler, saranno scelti come genitori femminile e maschile. Le piante devono essere giovani e sani. Si può evirare il genitore femminile utilizzando un microscopio da dissezione, visiera d&#39;ingrandimento, o occhio nudo. Individuare fase-12 fiori (Smyth et al., 1990) e rimuovere eventuali fiori o silique sopra e sotto di loro da taglio alla base del pedicello con le forbici. Sterilizzare pinze immergendo la base della punta delicatamente in un bicchiere di etanolo al 95%, che eliminerà qualsiasi grani di polline sulle pinze e uccidere il polline pure. Sollevare delicatamente a parte i boccioli dei fiori con pinze, e rimuovere delicatamente 4 sepali, 4 petali e 6 stami, lasciando il pistillo nudo e intatto. Cercate di evitare di danneggiare la carpelli durante questo processo. 2. La raccolta del donatore polline e lo svolgimento impollinazione I donatori migliori polline sono anthesed fiori aperti nella fase 14 con i petali si estende ad un angolo di 90 ° al pistillo (Smyth et al., 1990), in cui un sacco di polline è spargimento. Prendete il fiore alla base e appena sopra il peduncolo, che farà sì che il fiore di diffondere aperto. Polvere lo stigma del pistillo preparato con le antere. Dopo l&#39;impollinazione, lo stigma sarà coperto con il polline giallo, che può essere facilmente osservati al microscopio. 3. Etichettatura la croce Dopo impollinatori tutti i pistilli castrato su una pianta, si etichetta la croce con un tag gioielli e informazione dei genitori femminili e maschili e la data. Mettere un palo nel terreno vicino alla pianta, utilizzare una stringa per legare il fusto della infiorescenza al rogo, e coprire i pistilli impollinati con un sacchetto di plastica. II. Isolamento dei tessuti dell&#39;endosperma in Arabidopsis 1. Preparazione dei materiali Noi di solito ottengono materiali necessari pronto prima della raccolta della mandorla farinosa e tessuti dell&#39;embrione: un serbatoio di azoto liquido, azoto liquido, un microscopio da dissezione, due nuove coppie di fine-punta pinza (5 INOX FST da Dumont biologia, in Svizzera.), Vetrini microscopio ( 3 &quot;X 1&quot; X 1.0 mm), una soluzione di 5,7 pH di 0,3 M sorbitolo e 5 mM MES. 2. Silique raccolta A 7 - 8-o giorno dopo l&#39;impollinazione (DAP), i semi sono pronte per essere raccolte a metà e la fine della fase siluro embriogenesi e sezionato per endosperma ed embrione. A volte, potrebbe prendere 9-10 DAP se i fiori evirati sono troppo giovani. 3. Endosperma isolare ed embrione Dal momento che i semi di Arabidopsis sono piccoli, usiamo un microscopio da dissezione per isolare endosperma ed embrione. Mettere un 8-DAP Silique al microscopio, usare un paio di pinze per tenere il pedicello Silique e utilizzare la punta di un altro paio di pinze a scivolare aprire il Silique a margine dove fondono due carpelli. Usare un paio di pinze per prendere un seme sul pH 5,7 soluzioni di 0,3 M sorbitolo e 5 mM MES, fare un piccolo taglio alla fine micropilo a scivolare fuori embrione, spremere alla fine non tagliato per spingere fuori endosperma e endosperma separato da tegumento (Kinoshita et al., 2004). Mettere l&#39;embrione e endosperma in microtubi separati in azoto liquido. Continuare fino a quando questo si accumulano embrione o endosperma 10-15 silique e quindi memorizzare il tubo in un congelatore C ° -80. In alcuni casi, dalla mandorla farinosa non deve essere separato da tegumento, cioè, si può isolare l&#39;RNA totale da mandorla farinosa e del cappotto miscela di semi per l&#39;analisi del gene imprinting. III. Bisolfito Sequencing 1. Reagenti necessari Cetyltrimethyl ammonio bromuro (CTAB) per la preparazione di DNA genomico, enzimi di restrizione, 3 M di NaOH (appena fatto), 6,42 M urea / 4 bisolfito di sodio M (2 metabisolfito di sodio M, Sigma-Aldrich, S9000, Na2S2O5, peso molecolare: 190), 10 mM idrochinone, un kit di purificazione del DNA (Promega Wizard DNA Clean-up di sistema, cat. # A7280), TE buffer, 6,3 M di NaOH (appena fatto), 10 M NH 4 OAc, 20 mg / tRNA microlitri, e il 100% di etanolo . 2. I dettagli del protocollo di trattamento bisolfito <ol><li> Isolare endosperma del DNA genomico o embrione utilizzando una procedura CTAB (Rogers e Bendich, 1988). </li><li> Digest 100 ng - 2 mg di DNA genomico in 20 l per 100 l di volume totale con enzimi di restrizione che tagliano fuori della regione da analizzare. Per il promotore MEA, usiamo XhoI, NdeI e PstI o HindIII. </li><li> Denaturare gli enzimi di restrizione del DNA facendo bollire per cinque minuti e poi spegnete il ghiaccio. </li><li> Aggiunta del volume 09/01 (2,2 microlitri per 20 microlitri di DNA digerito) di 3 milioni di NaOH e incubare a 37 ° C per 15 minuti. </li><li> Trasferire la soluzione in una provetta da 250 microlitri PCR. </li><li> Sciogliere 7,5 g di urea in 10 ml di acqua distillata sterile; lentamente aggiungere 7,6 g di metabisolfito di sodio nell&#39;arco di 1-2 ore e il riscaldamento di solito aiuta la dissoluzione; Regolare il pH a 5 con appena fatto 10 M NaOH; Aggiungere acqua distillata sterile ad una finale volume a 20 mL. Si tratta di 6,24 M urea / 4 M bisolfito di sodio. </li><li> Aggiungi 6,24 M urea / 4 M bisolfito di sodio ad una concentrazione finale di 5,36 M e 3,44 M, rispettivamente (Paulin et al., 1998). Ad esempio, aggiungere 208 ml di 6,42 M urea / 4 M bisolfito di sodio al precedente 22,2 ml di DNA genomico denaturato (Xiao et al., 2003). </li><li> Aggiungere 10 mM idrochinone al DNA ad una concentrazione finale di 0,5 mm (12 microlitri per 20 digestione mL). </li><li> Condotta trattamento bisolfito in una macchina di PCR: 30 cicli di 55 ° C per 15 minuti e 95 ° C per 30 secondi. </li><li> Desalificare il DNA bisolfito trattato con il DNA guidata Clean-Up del sistema da Promega e seguire il protocollo (Jacobsen et al., 2000). </li><li> Misurare il volume esatto di TE recuperato dalla colonna dopo desalinizzazione e aggiungere 6,3 M di NaOH ad una concentrazione finale di 0,3 M. Incubare a 37 ° C per 15 minuti. </li><li> Aggiungere 10 M NH 4 OAc (pH 7,0) ad una concentrazione finale di 3 milioni, 2 ml di 20 mg / tRNA microlitri, e 3 volumi di etanolo 100% e poi mescolare. Centrifugare per 15 minuti a 14.000 giri al minuto. </li><li> Lavare il pellet una volta con etanolo al 70%, fare una centrifuga breve, ed eliminare l&#39;etanolo extra. </li><li> Secco pellet in un speedvac per 5-10 minuti e risospendere in 25-100 microlitri di buffer TE seconda partenza quantità di DNA. Il bisolfito di sodio-trattati DNA è ora pronto per l&#39;analisi PCR. </li></ol> 3. Amplificazione PCR <ol><li> Dal momento che non metilato cytosines vengono convertiti in uracile, è difficile per amplificare un frammento di grandi dimensioni utilizzando il bisolfito trattati con il DNA come un modello. Così, di solito disegno primer per amplificare un prodotto non più di 500 bp. Per la sequenza di 4 KB promotore MEA, abbiamo progettato molti set di primer e amplificati 14 frammenti sovrapposti per coprire l&#39;intera regione (Xiao et al., 2003). </li><li> Per la sequenza in alto a filo, nella progettazione di un primer forward, I) scegliere un (G guanina)-regione ricca in modo da avere una temperatura superiore di ricottura senza più nucleotidi nella primer; II) cambiare C (citosina) a Y ( pirimidina) in CG e contesti metano e cambiare il rimanente C a T (timina). Nella progettazione di un primer reverse, I) scegliere una regione ricca di C; II) cambiare G R (purine) in CG e contesti metano e cambiare il G rimanenti A (adenina). </li><li> Per la sequenza in basso a filo, nella progettazione di un primer forward, I), scegliere una regione ricca di C; II) cambiare G a R a CG e contesti metano e cambiare il G rimanenti ad A. Nella progettazione di un primer reverse, I) scegliere un G-regione ricca; II) C di variazione di Y (pirimidina) in CG e contesti metano e cambiare il rimanente C a T. </li><li> Usiamo solitamente 1-2 ml di DNA bisolfito di sodio, trattato come un modello per ogni amplificazione PCR (Xiao et al., 2003). Il prodotto di PCR deve essere analizzato mediante elettroforesi su gel per confermare la corretta dimensione del frammento, poi da gel purificato e clonato nel vettore di clonaggio TOPO TA pCR2.1 (Invitrogen) come inserto. Una singola colonia viene prelevato e colto; DNA plasmidico estratto e inviato per il sequenziamento. </li></ol> 4. Analisi di sequenze Il principio di sequenziamento bisolfito è che citosina non metilato verrà convertito in uracile a causa di deaminazione idrolitici da elevata concentrazione di bisolfito di sodio a pH5.0 che sarà amplificato come timina nel prodotto di PCR, mentre 5-metil citosina non verrà modificato da bisolfito di sodio e restano da citosina dopo amplificazione PCR (Clark et al, 1994;. Frommer et al, 1992).. Dopo aver ottenuto il risultato di sequenziamento, la confrontiamo con il filone specifico modello utilizzato per l&#39;amplificazione PCR. Se un residuo citosina nel modello si legge come una timina nel risultato di sequenziamento, indica che la citosina non è metilato. Se un residuo citosina nel modello rimane una citosina in sequenza, significa che la citosina è metilato. IV. Rappresentante Risultati <img src="/files/ftp_upload/2327/2327fig1.jpg" alt="Figura 1 /" /> Figura 1.

<ol>
	<li>Clark, S. J., Harrison, J., Paul, C. L., Frommer, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+sensitivity+mapping+of+methylated+cytosines.">High sensitivity mapping of methylated cytosines.</a> Nucleic Acids Res. 22, 2990-2997 (1994).</li><li>Cokus, S. J., Feng, S., Zhang, X., Chen, Z., Merriman, B., Haudenschild, C. D., Sriharsa Pradhan, S., Nelson, S. F., Pellegrini, M., Jacobsen, S. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shotgun+bisulphite+sequencing+of+the+Arabidopsis+genome+reveals+DNA+methylation+patterning.">Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning.</a> Nature. 452, 215-219 (2008).</li><li>Frommer, M., McDonald, L. E., Millar, D. S., Collis, C. M., Watt, F., Grigg, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genomic+sequencing+protocol+that+yields+a+positive+display+of+5-methylcytosine+residues+in+individual+DNA+strands.">A genomic sequencing protocol that yields a positive display of 5-methylcytosine residues in individual DNA strands.</a> Proc Natl Acad Sci USA. 89, 1827-1831 (1992).</li><li>Gehring, M., Huh, J. H., Hsieh, T. F., Penterman, J., Choi, Y., Harada, J. J., Goldberg, R. B., Fischer, R. L. D. E. M. E. T. E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=DNA+glycosylase+establishes+MEDEA+polycomb+gene+self-imprinting+by+allele-specific+demethylation.">DNA glycosylase establishes MEDEA polycomb gene self-imprinting by allele-specific demethylation.</a> Cell. 124, 495-506 (2006).</li><li>Henderson, I. R., Chan, S. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ectopic+hypermethylation+of+flower-specific+genes+in+Arabidopsis.">Ectopic hypermethylation of flower-specific genes in Arabidopsis.</a> Curr. Biol. 10, 179-186 (2000).</li><li>Kinoshita, T., Miura, A., Choi, Y., Kinoshita, Y., Cao, X., Jacobsen, S. E., Fischer, R. L., Kakutani, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-way+control+of+FWA+imprinting+in+Arabidopsis+endosperm+by+DNA+methylation.">One-way control of FWA imprinting in Arabidopsis endosperm by DNA methylation.</a> Science. 303, 521-523 (2004).</li><li>Lister, R., O'Malley, R. C., Tonti-Filippini, J., Gregory, B. D., Berry, C. C., Millar, A. H., Ecker, J. 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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Urea+improves+efficiency+of+bisulfite-mediated+sequencing+of+5'-+methylcytosine+in+genomic+DNA.">Urea improves efficiency of bisulfite-mediated sequencing of 5'- methylcytosine in genomic DNA.</a> Nucl. Acids Res. 26, 5009-5010 (1998).</li><li>Rogers, S. O., &amp;amp, B. e. n. d. i. c. h., J, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extraction+of+DNA+from+plant+tissues.">Extraction of DNA from plant tissues.</a> Plant Molecular Biology Manual. A6, 1-10 (1988).</li><li>Smyth, D. R., Bowman, J. L., Elliot, M., Meyerowitz, E. 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Nessun conflitto di interessi dichiarati.

E &#39;relativamente facile separare embrione da endosperma e tegumento, ma è noioso per separare endosperma da tegumento, soprattutto per le sementi in fase iniziale o medio-siluro di embriogenesi. Dal cappotto di seme contribuisce solo una piccola quantità di tessuto, per alcuni geni, ad esempio, MEA e FWA, non abbiamo a separare endosperma da tegumento. Questo significa che siamo in grado di isolare RNA da una miscela di endosperma e tessuti tegumento, controllare l&#39;espressione degli alleli ma...

Forniture <ul><li> Microscopio da dissezione </li><li> Forbici </li><li> Pinza punta fine </li><li> Gioielli Tag </li><li> Impianto Stakes </li><li> Stringa o Twist-Ties </li><li> 4 &quot;X 2&quot; X &quot;in polietilene 8 Borse </li><li> 3 &quot;X 1&quot; X preparati microscopici 1,0 millimetri </li><li> Azoto liquido </li><li> I contenitori di azoto liquido </li><li> Blocco di calore </li><li> Tubi di PCR </li><li> Termociclatore </li><li> Microcentrifuga Tubi </li><li> Microcentrifuga </li><li> Gel elettroforesi impianto </li><li> Arabidopsis thaliana Columbia-0 Piante </li><li> Arabidopsis thaliana Landsberg erecta Piante </li></ul> Reagenti <ul><li> 70% di etanolo </li><li> 95% di etanolo </li><li> 100% di etanolo </li><li> 0,3 M Sorbitolo e 5 mM MES-pH 5,7 </li><li> Cetyltrimethyl Bromuro di ammonio (CTAB) </li><li> 100% di etanolo </li><li> Cholorform </li><li> Gli enzimi di restrizione </li><li> 3 M NaOH </li><li> 6,3 M di NaOH </li><li> 6,24 M urea / 4 bisolfito di sodio M </li><li> Distillata sterile H 2 O </li><li> 10 mM Idrochinone </li><li> Wizard DNA Clean-Up System (Promega) </li><li> 10 M NH 4 OAc </li><li> 20 mg / tRNA microlitri </li><li> TE buffer di </li><li> Il TOPO TA Cloning Kit (Invitrogen) </li></ul>

determination of dna methylation of imprinted genes in arabidopsis endosperm

Arabidopsis thaliana è un organismo modello eccellente per lo studio dei meccanismi epigenetici. Uno dei motivi è la perdita di funzione mutante nullo di DNA metiltransferasi è vitale, fornendo così un sistema per studiare come la perdita di metilazione del DNA in un genoma influisce sulla crescita e lo sviluppo. Imprinting si riferisce alla espressione differenziale di alleli paterni e materni e svolge un ruolo importante nello sviluppo riproduzione in entrambi i mammiferi e piante. Metilazione del DNA è essenziale per determinare se gli alleli materni o paterni di un gene impresso è espressa o tacere. In piante da fiore, c&#39;è un evento doppia fecondazione in riproduzione: una cellula spermatozoo feconda l&#39;ovulo per formare embrione e un fusibili sperma secondo con la cella centrale per dar luogo a endosperma. Endosperma è il tessuto in cui imprinting si verifica nelle piante. MEDEA, un dominio gene SET Polycomb gruppo e FWA, un fattore di trascrizione che regolano la fioritura, sono i primi due geni dimostrato di essere impressa nella endosperma e la loro espressione è controllata da metilazione del DNA e demetilazione in piante. Al fine di determinare lo stato imprinting di un gene e pattern di metilazione in endosperma, dobbiamo essere in grado di isolare endosperma prima. Dal seme è piccolo in Arabidopsis, rimane difficile da isolare endosperma Arabidopsis ed esaminare la sua metilazione. In questo protocollo video, si segnala come condurre un incrocio, per isolare il tessuto dell&#39;endosperma da semi, e per determinare lo stato di metilazione mediante sequenziamento bisolfito.

Watch this Scientific Journal Video about Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm at JoVE.com

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

L&#39;imprinting è un fenomeno in impianti e riproduzione dei mammiferi. Metilazione del DNA gioca un ruolo importante nei meccanismi di imprinting. Isolando endosperma e determinare lo stato di metilazione dei geni impressa<em&gt; Arabidopsis</em&gt; Può essere difficile. In questo protocollo, si descrive come isolare endosperma e determinare metilazione mediante sequenziamento bisolfito.

Determinazione della metilazione del DNA di geni impressa Arabidopsis Dell&#39;endosperma

Arabidopsis thaliana is an excellent model organism for studying epigenetic mechanisms. One of the reasons is the loss-of-function null mutant of DNA ...

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Biology

Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

17.3K Views.  Saint Louis University. L'imprinting è un fenomeno in impianti e riproduzione dei mammiferi. Metilazione del DNA gioca un ruolo importante nei meccanismi di imprinting. Isolando endosperma e determinare lo stato di metilazione dei geni impressa Arabidopsis Può essere difficile. In questo protocollo, si descrive come isolare endosperma e determinare metilazione mediante sequenziamento bisolfito.

Video: Determination of DNA Methylation of Imprinted Genes in Arabidopsis Endosperm

DNA Methylation: Bisulphite Modification and Analysis

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene regulation is complex, but essentially involves modifications to the DNA itself or the proteins with which DNA associates. The predominant epigenetic modification of DNA in mammalian genomes is methylation of cytosine nucleotides (5-MeC). DNA methylation provides instruction to gene expression machinery as to where and when the gene should be expressed. The primary target sequence for DNA methylation in mammals is 5'-CpG-3' dinucleotides (Figure 1). CpG dinucleotides are not uniformly distributed throughout the genome, but are concentrated in regions of repetitive genomic sequences and CpG "islands" commonly associated with gene promoters (Figure 1). DNA methylation patterns are established early in development, modulated during tissue specific differentiation and disrupted in many disease states including cancer. To understand the biological role of DNA methylation and its role in human disease, precise, efficient and reproducible methods are required to detect and quantify individual 5-MeCs.
This protocol for bisulphite conversion is the "gold standard" for DNA methylation analysis and facilitates identification and quantification of DNA methylation at single nucleotide resolution. The chemistry of cytosine deamination by sodium bisulphite involves three steps (Figure 2). (1) Sulphonation: The addition of bisulphite to the 5-6 double bond of cytosine (2) Hydrolic Deamination: hydrolytic deamination of the resulting cytosine-bisulphite derivative to give a uracil-bisulphite derivative (3) Alkali Desulphonation: Removal of the sulphonate group by an alkali treatment, to give uracil. Bisulphite preferentially deaminates cytosine to uracil in single stranded DNA, whereas 5-MeC, is refractory to bisulphite-mediated deamination. Upon PCR amplification, uracil is amplified as thymine while 5-MeC residues remain as cytosines, allowing methylated CpGs to be distinguished from unmethylated CpGs by presence of a cytosine "C" versus thymine "T" residue during sequencing.
DNA modification by bisulphite conversion is a well-established protocol that can be exploited for many methods of DNA methylation analysis. Since the detection of 5-MeC by bisulphite conversion was first demonstrated by Frommer et al.1 and Clark et al.2, methods based around bisulphite conversion of genomic DNA account for the majority of new data on DNA methylation. Different methods of post PCR analysis may be utilized, depending on the degree of specificity and resolution of methylation required. Cloning and sequencing is still the most readily available method that can give single nucleotide resolution for methylation across the DNA molecule.

The gold standard for DNA methylation analysis is genomic sequencing of bisulphite converted DNA. This method takes advantage of the increased sensitivity of cytosine compared with 5-methylcytosine (5-MeC) to bisulphite deamination under acidic conditions. Unmethylated cytosines can be distinguished from methylated cytosines after PCR amplification of the target genomic DNA.

Metilazione del DNA: Modifica bisolfito e analisi

Epigenetics describes the heritable changes in gene function that occur independently to the DNA sequence. The molecular basis of epigenetic gene ...

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Biologia

Chromosomics: Detection of Numerical and Structural Alterations in All 24 Human Chromosomes Simultaneously Using a Novel OctoChrome FISH Assay

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell nuclei1. The technique uses DNA probes with unique sequences that hybridize to whole chromosomes or specific chromosomal regions, and serves as a powerful adjunct to classic cytogenetics. For instance, many earlier studies reported the frequent detection of increased chromosome aberrations in leukemia patients related with benzene exposure, benzene-poisoning patients, and healthy workers exposed to benzene, using classic cytogenetic analysis2. Using FISH, leukemia-specific chromosomal alterations have been observed to be elevated in apparently healthy workers exposed to benzene3-6, indicating the critical roles of cytogentic changes in benzene-induced leukemogenesis. 
Generally, a single FISH assay examines only one or a few whole chromosomes or specific loci per slide, so multiple hybridizations need to be conducted on multiple slides to cover all of the human chromosomes. Spectral karyotyping (SKY) allows visualization of the whole genome simultaneously, but the requirement for special software and equipment limits its application7. Here, we describe a novel FISH assay, OctoChrome-FISH, which can be applied for Chromosomics, which we define here as the simultaneous analysis of all 24 human chromosomes on one slide in human studies, such as chromosome-wide aneuploidy study (CWAS)8. The basis of the method, marketed by Cytocell as the Chromoprobe Multiprobe System, is an OctoChrome device that is divided into 8 squares, each of which carries three different whole chromosome painting probes (Figure 1). Each of the three probes is directly labeled with a different colored fluorophore, green (FITC), red (Texas Red), and blue (Coumarin). The arrangement of chromosome combinations on the OctoChrome device has been designed to facilitate the identification of the non-random structural chromosome alterations (translocations) found in the most common leukemias and lymphomas, for instance t(9;22), t(15;17), t(8;21), t(14;18)9. Moreover, numerical changes (aneuploidy) in chromosomes can be detected concurrently. The corresponding template slide is also divided into 8 squares onto which metaphase spreads are bound (Figure 2), and is positioned over the OctoChrome device. The probes and target DNA are denatured at high-temperature and hybridized in a humid chamber, and then all 24 human chromosomes can be visualized simultaneously. 
OctoChrome FISH is a promising technique for the clinical diagnosis of leukemia and lymphoma and for detection of aneuploidies in all chromosomes. We have applied this new Chromosomic approach in a CWAS study of benzene-exposed Chinese workers8,10.

A novel fluorescence in situ hybridization (FISH) method that simultaneously examines both numerical and structural chromosome alterations, particularly the specific chromosomal translocations associated with leukemia and lymphoma, of all 24 human chromosomes on a single device in one hybridization, is described.

Chromosomics: Rilevamento delle alterazioni numeriche e strutturali in tutti i 24 cromosomi umani simultaneamente utilizzando un nuovo metodo di analisi FISH OctoChrome

Fluorescence in situ hybridization (FISH) is a technique that allows specific DNA sequences to be detected on metaphase or interphase chromosomes in cell ...

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of dormant seeds or that of nondormant seeds irradiated by a far red (FR) light pulse. In order to further gain insight into the molecular genetic mechanisms underlying the germination repressive activity exerted by the endosperm, a &#34;seed coat bedding&#34; assay (SCBA) was devised. The SCBA is a dissection procedure physically separating seed coats and embryos from seeds, which allows monitoring the growth of embryos on an underlying layer of seed coats. Remarkably, the SCBA reconstitutes the germination repressive activities of the seed coat in the context of seed dormancy and FR-dependent control of seed germination. Since the SCBA allows the combinatorial use of dormant, nondormant and genetically modified seed coat and embryonic materials, the genetic pathways controlling germination and specifically operating in the endosperm and embryo can be dissected. Here we detail the procedure to assemble a SCBA.

A Seed Coat Bedding Assay to Genetically Explore In Vitro How the Endosperm Controls Seed Germination in Arabidopsis thaliana

We present the procedure to assemble a seed coat bedding assay (SCBA) from Arabidopsis thaliana seeds. The SCBA was shown to be a powerful tool to explore genetically and in vitro how the endosperm controls seed germination in dormant seeds and in response to light cues. The SCBA is in principle applicable under any situation where the endosperm is suspected to influence embryonic growth.

Un cappotto Seed Bedding Assay per esplorare geneticamente<em&gt; In Vitro</em&gt; Come la Germinazione Controlli endosperma del seme in<em&gt; Arabidopsis thaliana</em

The Arabidopsis endosperm consists of a single cell layer surrounding the mature embryo and playing an essential role to prevent the germination of ...

Real-time Imaging of Single Engineered RNA Transcripts in Living Cells Using Ratiometric Bimolecular Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the development of diverse techniques to visualize individual RNA transcripts in single living cells. One promising technique that has recently been described utilizes an oligonucleotide-based optical probe, ratiometric bimolecular beacon (RBMB), to detect RNA transcripts that were engineered to contain at least four tandem repeats of the RBMB target sequence in the 3&#8217;-untranslated region. RBMBs are specifically designed to emit a bright fluorescent signal upon hybridization to complementary RNA, but otherwise remain quenched. The use of a synthetic probe in this approach allows photostable, red-shifted, and highly emissive organic dyes to be used for imaging. Binding of multiple RBMBs to the engineered RNA transcripts results in discrete fluorescence spots when viewed under a wide-field fluorescent microscope. Consequently, the movement of individual RNA transcripts can be readily visualized in real-time by taking a time series of fluorescent images. Here we describe the preparation and purification of RBMBs, delivery into cells by microporation and live-cell imaging of single RNA transcripts.

Ratiometric bimolecular beacons (RBMBs) can be used to image single engineered RNA transcripts in living cells. Here, we describe the preparation and purification of RBMBs, delivery of RBMBs into cells by microporation and fluorescent imaging of single RNA transcripts in real-time.

In tempo reale Imaging del singolo Engineered RNA trascritti in cellule viventi Utilizzando Ratiometric bimolecolari Beacons

The growing realization that both the temporal and spatial regulation of gene expression can have important consequences on cell function has led to the ...

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation.

In vitro Methylation Assay to Study Protein Arginine Methylation

Protein arginine methylation, catalyzed by a class of enzymes viz., protein arginine methyl transferases (PRMTs), is the process of enzymatic addition of methyl group(s) to arginines within proteins. The in vitro methylation assay is the most dependable tool for assessing the methylation status of known or novel PRMT substrates.

In vitro metilazione Assay per studiare proteine ​​Arginina Metilazione

Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified ...

Enhanced Reduced Representation Bisulfite Sequencing for Assessment of DNA Methylation at Base Pair Resolution

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine methylation patterns in cells. Reduced representation of whole genome bisulfite sequencing was developed to detect quantitative base pair resolution cytosine methylation patterns at GC-rich genomic loci. This is accomplished by combining the use of a restriction enzyme followed by bisulfite conversion. Enhanced Reduced Representation Bisulfite Sequencing (ERRBS) increases the biologically relevant genomic loci covered and has been used to profile cytosine methylation in DNA from human, mouse and other organisms. ERRBS initiates with restriction enzyme digestion of DNA to generate low molecular weight fragments for use in library preparation. These fragments are subjected to standard library construction for next generation sequencing. Bisulfite conversion of unmethylated cytosines prior to the final amplification step allows for quantitative base resolution of cytosine methylation levels in covered genomic loci. The protocol can be completed within four days. Despite low complexity in the first three bases sequenced, ERRBS libraries yield high quality data when using a designated sequencing control lane. Mapping and bioinformatics analysis is then performed and yields data that can be easily integrated with a variety of genome-wide platforms. ERRBS can utilize small input material quantities making it feasible to process human clinical samples and applicable in a range of research applications. The video produced demonstrates critical steps of the ERRBS protocol.

Enhanced Reduced Representation Bisulfite Sequencing is a method for the preparation of sequencing libraries for DNA methylation analysis based on restriction enzyme digestion combined with cytosine bisulfite conversion. This protocol requires 50 ng of starting material and yields base pair resolution data at GC-rich genomic regions.

ENHANCED ridotto Rappresentanza bisolfito sequenziamento per la valutazione della metilazione del DNA in Risoluzione coppia di basi

DNA methylation pattern mapping is heavily studied in normal and diseased tissues. A variety of methods have been established to interrogate the cytosine ...

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a cytosine-guanine dinucleotide. While DNA methylation provides long-lasting and stable changes in gene expression, patterns and levels of DNA methylation are also subject to change based on a variety of signals and stimuli. As such, DNA methylation functions as a powerful and dynamic regulator of gene expression. The study of neuroepigenetics has revealed a variety of physiological and pathological states that are associated with both global and gene-specific changes in DNA methylation. Specifically, striking correlations between changes in gene expression and DNA methylation exist in neuropsychiatric and neurodegenerative disorders, during synaptic plasticity, and following CNS injury. However, as the field of neuroepigenetics continues to expand its understanding of the role of DNA methylation in CNS physiology, delineating causal relationships in regards to changes in gene expression and DNA methylation are essential. Moreover, in regards to the larger field of neuroscience, the presence of vast region and cell-specific differences requires techniques that address these variances when studying the transcriptome, proteome, and epigenome. Here we describe FACS sorting of cortical astrocytes that allows for subsequent examination of a both RNA transcription and DNA methylation. Furthermore, we detail a technique to examine DNA methylation, methylation sensitive high resolution melt analysis (MS-HRMA) as well as a luciferase promoter assay. Through the use of these combined techniques one is able to not only explore correlative changes between DNA methylation and gene expression, but also directly assess if changes in the DNA methylation status of a given gene region are sufficient to affect transcriptional activity.

Correlating Gene-specific DNA Methylation Changes with Expression and Transcriptional Activity of Astrocytic KCNJ10 Kir4.1

DNA methylation is capable of maintaining stable levels of gene expression as well as allowing for dynamic changes in gene expression in response to a variety of stimuli. We detail techniques that allow the study of gene-specific changes in DNA methylation and the effect of these changes on gene expression.

Correlare DNA Modifiche metilazione gene-specifica con espressione e trascrizionale attività di astrociti<em&gt; KCNJ10</em&gt; (Kir4.1)

DNA methylation serves to regulate gene expression through the covalent attachment of a methyl group onto the C5 position of a cytosine in a ...

Targeted DNA Methylation Analysis by Next-generation Sequencing

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of particular interest for epigenetic studies as it has been demonstrated to be both a long lasting and a dynamic regulator of gene expression. Efforts to examine epigenetic changes in health and disease have been hindered by the lack of high-throughput, quantitatively accurate methods. With the advent and popularization of next-generation sequencing (NGS) technologies, these tools are now being applied to epigenomics in addition to existing genomic and transcriptomic methodologies. For epigenetic investigations of cytosine methylation where regions of interest, such as specific gene promoters or CpG islands, have been identified and there is a need to examine significant numbers of samples with high quantitative accuracy, we have developed a method called Bisulfite Amplicon Sequencing (BSAS). This method combines bisulfite conversion with targeted amplification of regions of interest, transposome-mediated library construction and benchtop NGS. BSAS offers a rapid and efficient method for analysis of up to 10 kb of targeted regions in up to 96 samples at a time that can be performed by most research groups with basic molecular biology skills. The results provide absolute quantitation of cytosine methylation with base specificity. BSAS can be applied to any genomic region from any DNA source. This method is useful for hypothesis testing studies of target regions of interest as well as confirmation of regions identified in genome-wide methylation analyses such as whole genome bisulfite sequencing, reduced representation bisulfite sequencing, and methylated DNA immunoprecipitation sequencing.

Bisulfite amplicon sequencing (BSAS) is a method for quantifying cytosine methylation in targeted genomic regions of interest. This method uses bisulfite conversion paired with PCR amplification of target regions prior to next-generation sequencing to produce absolute quantitation of DNA methylation at a base-specific level.

Targeted DNA metilazione Analisi per sequenziamento di prossima generazione

The role of epigenetic processes in the control of gene expression has been known for a number of years. DNA methylation at cytosine residues is of ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Immunoprecipitazione della cromatina Saggio per la identificazione di Arabidopsis interazioni proteina-DNA<em&gt; In Vivo</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

Risoluzione Quantificazione alta del cristallino cellulosa accumulo in<em&gt; Arabidopsis</em&gt; Roots a Monitor tessuto-specifica delle cellule parete Modifiche

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...