Questo lavoro è stato supportato da assegni di ricerca di facoltà dall'ufficio il College delle arti e del preside di Scienze presso l'Università di Hartford.

Durante lo studio, trattamento e cura degli animali in osservanza politica National Institutes of Health, erano in conformità con le linee guida istituzionali e sono stati approvati dal comitato di uso e cura animali istituzionale degli animali Università di Hartford.
1. trascinamento degli animali
Nota: Venti settimana-vecchio maschio B6129SF2/J topi sono usati nello studio.
<ol><li>Salire sul treno topi per una 12 ore di luce (luce standard testa bianca) / 12h scuro ciclo per 2 settimane prima dell'esperimento. Nota: Qui tempo zeitgeber (ZT) 0 corrisponde a luci su e ZT12 a luci spente, mantenendo tutti gli altri fattori ambientali (cioè, alimento, acqua e temperatura ambiente) costante 21.</li>
</ol>2. preparazione di strumenti, terreno di coltura e sfida Medium
<ol><li>Autoclave pinze e le forbici per dissezione. Avvolgere le coppie di vetrini da microscopio smerigliato in foglio di alluminio e autoclave.</li>
	<li>Preparare mezzo di coltura con l'aggiunta di siero bovino fetale (FBS) di RPMI 1640 a una concentrazione finale di 10%. Preparare 10 mL di terreno di sfida in provette da 50 mL aggiungendo il seguente PAMPs al terreno di coltura (RPMI con 10% FBS); LPS (5 µ g/mL), ODN1826 (5 µ g/mL), calore-ucciso Listeria monocytogenes (108 HKLM/mL), o un altro PAMP nella sua concentrazione suggeriti.</li>
	<li>Caldo terreno di coltura e sfida medio a 37 ° C in un bagno d'acqua e tenere circa 70 mL di soluzione salina sterile tamponata al fosfato (PBS, pH 7,2) a temperatura ambiente.</li>
	<li>Aggiungere 10 mL di terreno di coltura utilizzando una pipetta da 10 mL e pipetta aiuti per un tubo da 50 mL e posto sul ghiaccio.</li>
	<li>Aggiungere circa 30 mL di etanolo al 70% a un'estremità di Becher e posto di 100ml di forbici e forcipe di dissezione nel bicchiere graduato per evitare la contaminazione microbica.</li>
	<li>Preparare il tampone di lisi per isolamento del RNA aggiungendo 10 µ l di β-mercaptoetanolo (sotto cappa aspirante) per ogni 1 mL di Buffer RLT in una provetta da 50 mL. Fare solo l'importo che sarà necessario (600 µ l per campione, che consiste di circa 1 x 106 cellule). Nota: Il Buffer RLT è un componente del kit di estrazione di RNA che supporta l'associazione di RNA per la membrana di silice.</li>
</ol>3. Splenocyte isolamento e sfida
<ol><li>Eutanasia topi in un momento di particolare zeitgeber tramite narcosi mantenendoli nella loro gabbia originale e aggiungendo CO2 alla gabbia ad una portata di 3 L/min continua fornitura di CO2 per 1 min dopo l'arresto della respirazione.</li>
	<li>Confermare la morte tramite dislocazione cervicale posizionando il pollice e il dito indice su entrambi i lati del collo alla base del cranio. In alternativa, premere una canna alla base del cranio mentre si tira rapidamente (con l'altra mano) la base della coda o le zampe per causare la separazione delle vertebre cervicali dal cranio22.</li>
	<li>Spruzzare il tronco del mouse con etanolo al 70% e asciugare con un tovagliolo di carta. Posizionare il mouse sulla sua schiena e leggermente inclinata sul fianco destro. Tagliare via la pelliccia, mediante dissezione Forbici, lato sinistro del mouse, circa a metà strada tra la parte anteriore e le zampe posteriori.</li>
	<li>Usando il forcipe, afferrare il peritoneo e incidete con attenzione per non danneggiare la milza. Rimuovere la milza con le pinzette e posto in una provetta sterile da 50 mL contenente circa 10 mL di terreno di coltura su ghiaccio. Ripetere per gli altri animali. Nota: La milza è il colore di un fagiolo, ed è più lungo e più piatta rispetto il rene.</li>
	<li>
Trasferire una milza con 2 mL di terreno di coltura per un piccolo piatto di Petri sterile.
	<ol>
<li>Omogeneizzare la milza di macinandolo fra la parte glassata di due diapositive smerigliati sterile. Se possibile, mantenere il problema e le cellule nel medium durante il processo di omogeneizzazione.</li>
		<li>Una volta accuratamente omogeneizzato, pipettare 2 mL di terreno di coltura contenente gli splenocytes attraverso un colino di cella di 40 µm in nylon in una provetta da 50 mL.</li>
		<li>Ripetere i passaggi sopra riportati per le milze rimanenti utilizzando nuove coppie di diapositive smerigliati, provette da 50 mL con terreno di coltura e filtri di cella.</li>
	</ol>
</li>
	<li>Aggiungere 8 mL di terreno di coltura fredda a ciascuna delle provette 50ml contenente gli splenocytes per un volume totale di 10 mL/tubo. Determinare il numero di cellule per millilitro utilizzando un emocitometro.</li>
	<li>Aggiungere circa 1 x 106 cellule/pozzetto in piastre di coltura 6 pozzetti. Aggiungere celle per il numero di pozzetti che corrispondono al numero di diversi PAMPs viene utilizzato per l'esperimento e includere un controllo bene. Aggiungere 3 mL di terreno di coltura o 3 mL di terreno di sfida a tutti i pozzetti.</li>
	<li>Incubare le piastre a 37 ° C in 5% CO2 per 3 h.</li>
	<li>Raschiare le cellule dal fondo del bene usando un puntale da 1.000 µ l collegato a una micropipetta P1000. Trasferire il supporto che contiene le celle utilizzando la stessa punta di dispensare 1.000 µ l in una provetta da 15 mL.</li>
	<li>Agglomerare le cellule tramite centrifugazione a 167 x g per 5 min a temperatura ambiente. Eliminare il surnatante e lavare il pellet cellulare con 5 mL di PBS.</li>
	<li>Agglomerare le cellule una seconda volta a 167 x g per 5 min, rimuovere il supernatante e aggiungere 600 µ l di tampone di lisi per il pellet cellulare per lisare le cellule. Procedere all'isolamento del RNA.</li>
</ol>4. isolamento RNA e la sintesi del cDNA
<ol><li>Isolare il RNA da splenocytes utilizzando il kit di estrazione di RNA secondo le istruzioni del produttore ed eseguire la digestione di DNA in colonna 'optional'.</li>
	<li>Prima sintesi del cDNA, calcolare la concentrazione di RNA mediante uno spettrofotometro di microvolume per verificare che la concentrazione sia all'interno della gamma ottima di RNA (fino a 2 µ g) di kit di sintesi di cDNA.</li>
	<li>
Synthesize cDNA per ciascuno dei campioni utilizzando il kit di trascrizione inversa secondo le istruzioni del produttore. Utilizzare 10 µ l di RNA per ognuno dei campioni in un volume di reazione totale di 20 µ l. Al completamento della trascrizione inversa (thermocycler esegue) aggiungere 20 μL H2O ad ogni reazione.
</li>
	<li>Costruire la curva standard utilizzando una micropipetta P10 o P20 per piscina mRNA da alcuni campioni di controllo (ad esempio, 5 µ l da 2 campioni per un totale di 10 µ l) in una provetta 0,5 mL per preparare il cDNA.<ol>
<li>Aggiungere 10 µ l 2 x mix master della trascrittasi inversa.</li>
		<li>Al completamento della trascrizione inversa, aggiungere 10 µ l di H2O al tubo di reazione, che servirà come la concentrazione iniziale ("1") della serie di diluizione per la curva standard.</li>
		<li>Eseguire una serie di diluizioni 10 volte (da 1 a 10-4) con l'aggiunta di 45 µ l di acqua utilizzando una micropipetta P100 o P200 nelle quattro provette 0,5 mL indicate 10-1, 10-2, 10-3e 10-4.<ol>
<li>Aggiungere 5 µ l di concentrazione iniziale "1" nel primo tubo (10-1) usando una micropipetta P20, mix pipettando su e giù parecchie volte, quindi trasferire 5 µ l dalla provetta 10-1 nel tubo indicato 10-2 e mix.</li>
			<li>Trasferire 5 µ l utilizzando una micropipetta P20 dal 10-2 tubo nel tubo indicato 10-3 e mescolare. Trasferire 5 µ l usando una micropipetta P20 dal 10-3 tubo nel tubo indicato 10-4 e mescolare.</li>
		</ol>
</li>
	</ol>
</li>
</ol>5. quantitativo reazione a catena della polimerasi (qPCR)
<ol><li>Determinare la quantificazione relativa dei livelli di mRNA di qPCR. All'interno l'allestimento sperimentale, selezionare "Quantificazione – relativa curva Standard" e "TaqMan" chimica. Cambiare il volume di reazione a 10 µ l. Immettere le informazioni rilevanti nel layout del piatto (ad es., gene bersaglio, curva standard, giornalista, ecc.).</li>
	<li>Preparare la reazione, in modo che esso contiene 0,5 µ l del dosaggio primer/sonda, 5 µ l di mix master di espressione genica, 2 µ l di H2O e 2,5 µ l di cDNA (10 – 100 ng).<ol>
<li>Preparare un mix master moltiplicando ciascuno dei reagenti precedenti (tranne il cDNA) per il numero di reazioni, assicurandosi di includere quelli dalla curva standard, controlli negativi, esibendosi ogni reazione in duplicato e 2 reazioni supplementare che rappresenteranno per variazione di pipettaggio.</li>
		<li>Dispensare 7,5 µ l del mix master preparati in pre-determinati pozzetti in una piastra di reazione a 96 pozzetti con una micropipetta o una pipetta multicanale. Dispensare 2,5 µ l di cDNA nel pozzo appropriato per le incognite e gli standard e 2,5 µ l di H2O nei pozzetti di controllo negativo.</li>
		<li>Sono saggi di primer/sonda per vari geni orologio molecolare e includono un'analisi per un controllo endogeno (ad es., β-actina).</li>
	</ol>
</li>
	<li>Al fine di determinare i valori relativi di espressione, calcolare la quantità relativa media per ogni replica, quindi, per ogni campione, dividere la quantità relativa media per il gene dell'obiettivo di quantità relativa media per β-actina.</li>
</ol>6. elaborazione statistica
<ol><li>Utilizzando software di analisi statistica, inserire i dati nel programma sotto l'opzione di statistiche di colonna. Selezionare un one-way ANOVA con il test post-hoc di Dunnett per valutare le differenze tra i valori medi di espressione del gene dell'orologio dopo la sfida PAMP contro il controllo.</li>
</ol>

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L'autore non ha nulla di divulgare.

All'interno di questo protocollo, uno spettrofotometro di microvolume può essere utilizzato per quantificare e valutare la purezza del RNA utilizzato nel determinare l'espressione genica. Acidi nucleici assorbono UV luce a 260 nm, proteine in genere assorbono la luce a 280 nm, mentre altri potenziali contaminanti utilizzati durante una procedura di estrazione di RNA (ad es., fenolo) sono rilevabili a 230 nm. Pertanto, valutando l'assorbanza (A) rapporto a 260/280 nm (RNA alla proteina) e 260/230 nm (RNA a prote...

Il master clock nei mammiferi, che orchestra oscillazioni 24h per quasi tutti gli aspetti della fisiologia e del comportamento, si trova all'interno del nucleo soprachiasmatico (SCN) dell'ipotalamo1,2. Oltre alla regolazione di processi biologici a livello organico, il master clock sincronizza anche periferici Orologi Cellulari in tutto il corpo3,4,5. Mentre il macchinario di orologio molecolare è costituito da almeno tre cicli di feedback trascrizionale traslazionale ad incastro, il nucleo è composto il periodo (Per1-3), crittocromo (Cry1-2), Bmal1, e orologio geni6,7. Oltre a mantenere la tempistica accurata dell'orologio molecolare core, anche alcuni prodotti del gene orologio accessorie (ad es., Rev-erbα e Dbp) regolano l'espressione dei geni dell'orologio non, cioè, orologio geni controllati (GCC)6 , 7.
Orologi molecolari funzionali sono state descritte in vari tessuti immuni (ad es., milza e linfonodi)8 e cellule (ad es., B cellule, cellule dendritiche, macrofagi)8,9. Queste cellule rilevare e rispondere ai pattern molecolari associati (PAMPs), conservati componenti microbici, attraverso recettori di riconoscimento immune innata come recettori Toll-like (TLR)10. Ad oggi, sono stati descritti 13 funzionale TLR, che riconoscono costituenti microbici quali componenti della parete cellulare batterica, proteina fustigatrice e acidi nucleici microbici10. La PAMP, lipopolisaccaride (LPS), un componente della parete cellulare dei batteri gram-negativi, riconosciuto da TLR4, è stato indicato per alterare i ritmi circadiani a livello sia organismica e molecolare. Ad esempio, in vivo sfida di LPS indotto da ritardi di fase fotica-come misurata dall'attività in topi11 e portato a orologio ridotta espressione genica nel SCN e nel fegato come determinato mediante ibridazione in situ e PCR quantitativa, rispettivamente, in ratti12. Dopo una sfida in vivo con LPS, analisi del sangue periferico umano leucociti13 e tessuto adiposo sottocutaneo14 ha rivelato alterata espressione di diversi geni dell'orologio come misurato tramite qPCR. Infine, ex vivo sfide LPS di macrofagi umani e macrofagi peritoneali del topo, inoltre ha condotto ad alterata espressione dell'orologio come misurato da qPCR14.
Qui, descriviamo un protocollo per valutare l'influenza dei LPS PAMPs, ODN1826 (oligonucleotidi sintetici contenenti unmethylated CpG motivi) e calore-ucciso Listeria monocytogenes (HKLM), riconosciuto da TLR4, TLR9 e TLR2, rispettivamente, il espressione genica di orologio molecolare negli splenocytes del topo. Il protocollo comprende lo splenectomy mouse, isolamento splenocyte e sfida, RNA estrazione, sintesi del cDNA e qPCR per valutare l'espressione di diversi geni dell'orologio. Questo protocollo permette l'acquisizione tempestiva di un gran numero di cellule del sistema immunitario con molto piccolo animale o manipolazione cellulare, che può quindi essere contestato ex vivo con vari PAMPs. L'orologio molecolare è stato indicato per modulare i vari aspetti della risposta immunitaria8,15,16, pertanto, rottura dell'orologio molecolare molto probabilmente potrebbe compromettere la corretta variazione dipendente dal tempo della risposta immunitaria. Inoltre, poiché le interruzioni dei ritmi circadiani possono portare a gravi patologie17,18,19,20, può essere di interesse per i ricercatori di sfidare gli splenocytes con una vasta gamma di molecole e valutare la loro influenza sull'orologio.

<table border="0" cellpadding="0" cellspacing="0" style="width:796px;" width="795">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="20">
			<td height="20" style="height:20px;width:343px;">Frosted slides</td>
			<td style="width:116px;">Fisher</td>
			<td style="width:137px;">12-550-343</td>
			<td style="width:200px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Cell strainers</td>
			<td>Fisher</td>
			<td align="right">22363547</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Lipopolysaccharide&#160;</td>
			<td>InvivoGen</td>
			<td>ltrl-eklps</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">ODN1826</td>
			<td>InvivoGen</td>
			<td>Tlrl-1826-1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HKLM</td>
			<td>InvivoGen</td>
			<td>Tlrl-hklm</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RPMI 1640</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">PBS</td>
			<td>Gibco</td>
			<td>20012-043</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNeasy Mini Kit</td>
			<td>Qiagen</td>
			<td>74104 or 74106</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">RNase-Free DNase Set</td>
			<td>Qiagen</td>
			<td align="right">79254</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-well cell culture plate</td>
			<td>Denville</td>
			<td>T1006</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">50 ml tubes</td>
			<td>Corning</td>
			<td align="right">352070</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">15 ml tubes</td>
			<td>Corning</td>
			<td align="right">352097</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High Capacity cDNA Reverse Transcription Kit</td>
			<td>ThermoFisher</td>
			<td align="right">4368814</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays b-actin</td>
			<td>ThermoFisher</td>
			<td>Mm00607939_s1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Per2</td>
			<td>ThermoFisher</td>
			<td>Mm00478113_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Rev-erba</td>
			<td>ThermoFisher</td>
			<td>Mm00520708_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Bmal1</td>
			<td>ThermoFisher</td>
			<td>Mm00500226_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Assays Dbp</td>
			<td>ThermoFisher</td>
			<td>Mm00497539_m1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">qPCR machine StepOnePlus</td>
			<td>ThermoFisher</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">TaqMan Gene Expression Master Mix</td>
			<td>ThermoFisher</td>
			<td align="right">4369016</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">MicroAmp Fast 96-well reaction plate (0.1 ml)</td>
			<td>ThermoFisher</td>
			<td align="right">4346907</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Statistical Analysis Software</td>
			<td>Prism 7.0a</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

the use of mouse splenocytes to assess pathogen-associated molecular pattern influence on clock gene expression

Dal comportamento di espressione genica, i ritmi circadiani regolano quasi tutti gli aspetti della fisiologia. Qui, presentiamo una metodologia per sfidare splenocytes del topo con il pattern molecolari associati (PAMPs) lipopolysaccharide (LPS), il ODN1826 e il calore-ucciso Listeria monocytogenes ed esaminare il loro effetto su molecolare circadiano orologio. In precedenza, studi si sono concentrati sull'esame l'influenza di LPS sull'orologio molecolare usando una varietà di approcci in vivo ed ex vivo da un assortimento di modelli (ad es., topo, ratto e umani). Questo protocollo descrive l'isolamento e la sfida di splenocytes, nonché la metodologia per valutare la post-sfida espressione di gene di orologio tramite PCR quantitativa. Questo approccio può essere utilizzato per valutare non solo l'influenza di componenti microbiche sull'orologio molecolare ma anche altre molecole che può alterare l'espressione dell'orologio. Questo approccio potrebbe essere utilizzato per prendere in giro a pezzi il meccanismo molecolare di come l'interazione di PAMP-Toll-like receptor influenza espressione dell'orologio.

Topi sono stati sacrificati a ZT13, splenocytes sono stati isolati e contestato ex vivo con il LPS PAMPs, ODN1826 o HKLM. Dopo 3 h, RNA è stato isolato e qPCR è stato utilizzato per valutare i livelli di espressione relativa dei geni orologio molecolare orologio, Per2, Dbp e Rev-erbα rispetto alle cellule di controllo incontrastato. Dopo la sfida PAMP, i livelli di espressione di Clock non erano significativamente differenti che l'e...

Watch this Scientific Journal Video about The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression at JoVE.com

The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Questo protocollo descrive una tecnica usando splenocytes del topo per scoprire pattern molecolari associati che alterano l'espressione genica di orologio molecolare.

L'uso del Mouse Splenocytes per valutare Pathogen-associated Molecular Pattern influenza sull'espressione del Gene dell'orologio

From behavior to gene expression, circadian rhythms regulate nearly all aspects of physiology. Here, we present a methodology to challenge mouse ...

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7.4K Views.  University of Hartford. Questo protocollo descrive una tecnica usando splenocytes del topo per scoprire pattern molecolari associati che alterano l'espressione genica di orologio molecolare.

Video: The Use of Mouse Splenocytes to Assess Pathogen-associated Molecular Pattern Influence on Clock Gene Expression

Parallel Measurement of Circadian Clock Gene Expression and Hormone Secretion in Human Primary Cell Cultures

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental changes. Considerable progress in our understanding of the tight connection between the circadian clock and most aspects of physiology has been made in the field over the last decade. However, unraveling the molecular basis that underlies the function of the circadian oscillator in humans stays of highest technical challenge. Here, we provide a detailed description of an experimental approach for long-term (2-5 days) bioluminescence recording and outflow medium collection in cultured human primary cells. For this purpose, we have transduced primary cells with a lentiviral luciferase reporter that is under control of a core clock gene promoter, which allows for the parallel assessment of hormone secretion and circadian bioluminescence. Furthermore, we describe the conditions for disrupting the circadian clock in primary human cells by transfecting siRNA targeting CLOCK. Our results on the circadian regulation of insulin secretion by human pancreatic islets, and myokine secretion by human skeletal muscle cells, are presented here to illustrate the application of this methodology. These settings can be used to study the molecular makeup of human peripheral clocks and to analyze their functional impact on primary cells under physiological or pathophysiological conditions.

Here, we describe settings to monitor in parallel circadian bioluminescence and the secretory activity of human islet cells and primary myotubes. For this, we employed lentiviral gene delivery of a luciferase core clock reporter, followed by in vitro synchronization and collection of outflow medium by continuous cell perifusion.

Misura parallela di circadiano Clock espressione genica e la secrezione dell&#39;ormone in colture cellulari primarie umane

Circadian clocks are functional in all light-sensitive organisms, allowing for an adaptation to the external world by anticipating daily environmental ...

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Single-cell Gene Expression Using Multiplex RT-qPCR to Characterize Heterogeneity of Rare Lymphoid Populations

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell activation, stress, or stimulation. Single-cell multiplex gene expression enables the simultaneous assessment of tens of genes1,2,3. At the single-cell level, multiplex gene expression determines population heterogeneity4,5. It allows for the distinction of population heterogeneity by determining both the probable mix of diverse precursor stages among mature cells and also the diversity of cell responses to stimuli.
Innate lymphoid cells (ILC) have been recently described as a population of innate effectors of the immune response6,7. In this protocol, cell heterogeneity of the ILC hepatic compartment is investigated during homeostasis.
Currently, the most widely used technique to assess gene expression is RT-qPCR. This method measures gene expression only one gene at a time. Additionally, this method cannot estimate heterogeneity of gene expression, since multiple cells are needed for one test. This leads to the measurement of the average gene expression of the population. When assessing large numbers of genes, RT-qPCR becomes a time-, reagent-, and sample-consuming method. Hence, the trade-offs limit the number of genes or cell populations that can be evaluated, increasing the risk of missing the global picture.
This manuscript describes how single-cell multiplex RT-qPCR can be used to overcome these limitations. This technique has benefited from recent microfluidics technological advances1,2. Reactions occurring in multiplex RT-qPCR chips do not exceed the nanoliter-level. Hence, single-cell gene expression, as well as simultaneous multiple gene expression, can be performed in a reagent-, sample-, and cost-effective manner. It is possible to test cell gene signature heterogeneity at the clonal level between cell subsets within a population at different developmental stages or under different conditions4,5. Working on rare populations with large numbers of conditions at the single-cell level is no longer a restriction.

This protocol describes how to assess the expression of a large array of genes at the clonal level. Single-cell RT-qPCR produces highly reliable results with a strong sensitivity for hundreds of samples and genes.

Unicellulare espressione genica Utilizzando Multiplex RT-qPCR per caratterizzare eterogeneità delle rare popolazioni linfoidi

Gene expression heterogeneity is an interesting feature to investigate in lymphoid populations. Gene expression in these cells varies during cell ...

Studying Cell Cycle-regulated Gene Expression by Two Complementary Cell Synchronization Protocols

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such as cancer. Cell cycle-regulated gene expression analysis depends on cell synchronization into specific phases. Here we describe a method utilizing two complementary synchronization protocols that is commonly used for studying periodic variation of gene expression during the cell cycle. Both procedures are based on transiently blocking the cell cycle in one defined point. The synchronization protocol by hydroxyurea (HU) treatment leads to cellular arrest in late G1/early S phase, and release from HU-mediated arrest provides a cellular population uniformly progressing through S and G2/M. The synchronization protocol by thymidine and nocodazole (Thy-Noc) treatment blocks cells in early mitosis, and release from Thy-Noc mediated arrest provides a synchronized cellular population suitable for G1 phase and S phase-entry studies. Application of both procedures requires monitoring of the cell cycle distribution profiles, which is typically performed after propidium iodide (PI) staining of the cells and flow cytometry-mediated analysis of DNA content. We show that the combined use of two synchronization protocols is a robust approach to clearly determine the transcriptional profiles of genes that are differentially regulated in the cell cycle (i.e. E2F1 and E2F7), and consequently to have a better understanding of their role in cell cycle processes. Furthermore, we show that this approach is useful for the study of mechanisms underlying drug-based therapies (i.e. mitomycin C, an anticancer agent), because it allows to discriminate genes that are responsive to the genotoxic agent from those solely affected by cell cycle perturbations imposed by the agent.

We report two cell synchronization protocols that provide a context for studying events related to specific phases of the cell cycle. We show that this approach is useful for analyzing the regulation of specific genes in an unperturbed cell cycle or upon exposure to agents affecting the cell cycle.

Studiare l&#39;espressione del gene regolamentata mediante ciclo cellulare da due protocolli complementari di sincronizzazione delle cellule

The gene expression program of the cell cycle represents a critical step for understanding cell cycle-dependent processes and their role in diseases such ...

Sample Preparation and Analysis of RNASeq-based Gene Expression Data from Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an excellent model for rapid assessment of whole transcriptome from whole animal or individual cell populations due to the ease of isolation of RNA from large numbers of animals. Here a protocol for global gene expression analysis in zebrafish embryos using RNA sequencing (RNASeq) is presented. We describe preparation of RNA from whole embryos or from cell populations obtained using cell sorting in transgenic animals. We also describe an approach for analysis of RNASeq data to identify enriched pathways and Gene Ontology (GO) terms in global gene expression data sets. Finally, we provide a protocol for validation of gene expression changes using quantitative reverse transcriptase PCR (qRT-PCR). These protocols can be used for comparative analysis of control and experimental sets of zebrafish to identify novel gene expression changes, and provide molecular insight into phenotypes of interest.

This protocol presents an approach for whole transcriptome analysis from zebrafish embryos, larvae, or sorted cells. We include isolation of RNA, pathway analysis of RNASeq data, and qRT-PCR-based validation of gene expression changes.

Preparazione dei campioni e analisi dei dati di espressione genica basati su RNASeq dallo Zebrafish

The analysis of global gene expression changes is a valuable tool for identifying novel pathways underlying observed phenotypes. The zebrafish is an ...

A Standard Methodology to Examine On-site Mutagenicity As a Function of Point Mutation Repair Catalyzed by CRISPR/Cas9 and SsODN in Human Cells

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point mutations, which often are responsible for a variety of human inherited disorders. Using a well-established cell-based model system, the point mutation of a single-copy mutant eGFP gene integrated into HCT116 cells has been repaired using this combinatorial approach. The analysis of corrected and uncorrected cells reveals both the precision of gene editing and the development of genetic lesions, when indels are created in uncorrected cells in the DNA sequence surrounding the target site. Here, the specific methodology used to analyze this combinatorial approach to the gene editing of a point mutation, coupled with a detailed experimental strategy to measuring indel formation at the target site, is outlined. This protocol outlines a foundational approach and workflow for investigations aimed at developing CRISPR/Cas9-based gene editing for human therapy. The conclusion of this work is that on-site mutagenesis takes place as a result of CRISPR/Cas9 activity during the process of point mutation repair. This work puts in place a standardized methodology to identify the degree of mutagenesis, which should be an important and critical aspect of any approach destined for clinical implementation.

This protocol outlines the workflow of a CRISPR/Cas9-based gene editing system for the repair of point mutations in mammalian cells. Here, we use a combinatorial approach to gene editing with a detailed follow-on experimental strategy for measuring indel formation at the target site&#8212;in essence, analyzing onsite mutagenesis.

Una metodologia Standard per esaminare in loco mutagenicità in funzione della mutazione di punto riparazione catalizzata da CRISPR/Cas9 e SsODN in cellule umane

Combinatorial gene editing using CRISPR/Cas9 and single-stranded oligonucleotides is an effective strategy for the correction of single-base point ...

Production and Purification of Baculovirus for Gene Therapy Application

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a promising vector for gene therapy application. Here, baculovirus is produced by transient transfection of the baculovirus plasmid DNA (bacmid) in an adherent culture of Sf9 cells. Baculovirus is subsequently expanded in Sf9 cells in a serum-free suspension culture until the desired volume is obtained. It is then purified from the culture supernatant using heparin affinity chromatography. Virus supernatant is loaded onto the heparin column which binds baculovirus particles in the supernatant due to the affinity of heparin for baculovirus envelop glycoprotein. The column is washed with a buffer to remove contaminants and baculovirus is eluted from the column with a high-salt buffer. The eluate is diluted to an isotonic salt concentration and baculovirus particles are further concentrated using ultracentrifugation. Using this method, baculovirus can be concentrated up to 500-fold with a 25% recovery of infectious particles. Although the protocol described here demonstrates the production and purification of the baculovirus from cultures up to 1 L, the method can be scaled-up in a closed-system suspension culture to produce a clinical-grade vector for gene therapy application.

In this protocol, baculovirus is produced by transient transfection of baculovirus plasmid into Sf9 cells and amplified in a serum-free suspension culture. The supernatant is purified by heparin affinity chromatography and further concentrated by ultracentrifugation. This protocol is useful for the production and purification of baculovirus for gene therapy application.

Produzione e purificazione di Baculovirus per applicazione di terapia genica

Baculovirus has traditionally been used for the production of recombinant protein and vaccine. However, more recently, baculovirus is emerging as a ...

An Optogenetic Method to Control and Analyze Gene Expression Patterns in Cell-to-cell Interactions

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling pathway is one of such essential molecular machinery for cell-to-cell communications, which plays key roles in normal development of embryos. This pathway involves a cell-to-cell transfer of oscillatory information with ultradian rhythms, but despite the progress in molecular biology techniques, it has been challenging to elucidate the impact of multicellular interactions on oscillatory gene dynamics. Here, we present a protocol that permits optogenetic control and live monitoring of gene expression patterns in a precise temporal manner. This method successfully revealed that intracellular and intercellular periodic inputs of Notch signaling entrain intrinsic oscillations by frequency tuning and phase shifting at the single-cell resolution. This approach is applicable to the analysis of the dynamic features of various signaling pathways, providing a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Here, we present a protocol to analyze cell-to-cell transfer of oscillatory information by optogenetic control and live monitoring of gene expression. This approach provides a unique platform to test a functional significance of dynamic gene expression programs in multicellular systems.

Un metodo di optogenetica per controllare e analizzare il pattern di espressione genica nelle interazioni della cellula--cellula

Cells should respond properly to temporally changing environments, which are influenced by various factors from surrounding cells. The Notch signaling ...

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3&#39; region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

In the present study, we describe a methodology to analyze the C924T genotype. The protocol consists of three phases: DNA extraction, amplification by polymerase chain reaction (PCR), and analysis of the restriction fragment length polymorphism (RFLP) on agarose gel.

Un metodo per studiare il polimorfismo C924T del Gene del ricevitore di trombossano A2

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in ...

Using Mouse Oocytes to Assess Human Gene Function During Meiosis I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively correlated with maternal age, age alone is not always predictive of the risk of generating an aneuploid embryo. Therefore, gene variants might account for incorrect chromosome segregation during oogenesis. Given that access to human oocytes is limited for research purposes, a series of assays were developed to study human gene function during meiosis I using mouse oocytes. First, messenger RNA (mRNA) of the gene and gene variant of interest are microinjected into prophase I-arrested mouse oocytes. After allowing time for expression, oocytes are synchronously released into meiotic maturation to complete meiosis I. By tagging the mRNA with a sequence of a fluorescent reporter, such as green fluorescent protein (Gfp), the localization of the human protein can be assessed in addition to the phenotypic alterations. For example, gain or loss of function can be investigated by establishing experimental conditions that challenge the gene product to fix meiotic errors. Although this system is advantageous in investigating human protein function during oogenesis, adequate interpretation of results should be undertaken given that protein expression is not at endogenous levels and, unless controlled for (i.e. knocked out or down), murine homologs are also present in the system.

As the genetic variants associated with human disease begin to become uncovered, it is becoming increasingly important to develop systems with which to rapidly evaluate the biological significance of those identified variants. This protocol describes methods for evaluating human gene function during female meiosis I using mouse oocytes.

Usando i Oocytes del Mouse per valutare la funzione del Gene umano durante la meiosi I

Embryonic aneuploidy is the major genetic cause of infertility in humans. Most of these events originate during female meiosis, and albeit positively ...

Exploring the Root Microbiome: Extracting Bacterial Community Data from the Soil, Rhizosphere, and Root Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to abiotic stresses and diseases. As the plant microbiome can be highly complex, low-cost, high-throughput methods such as amplicon-based sequencing of the 16S rRNA gene are often preferred for characterizing its microbial composition and diversity. However, the selection of appropriate methodology when conducting such experiments is critical for reducing biases that can make analysis and comparisons between samples and studies difficult. This protocol describes in detail a standardized methodology for the collection and extraction of DNA from soil, rhizosphere, and root samples. Additionally, we highlight a well-established 16S rRNA amplicon sequencing pipeline that allows for the exploration of the composition of bacterial communities in these samples, and can easily be adapted for other marker genes. This pipeline has been validated for a variety of plant species, including sorghum, maize, wheat, strawberry, and agave, and can help overcome issues associated with the contamination from plant organelles.

Here, we describe a protocol to obtain amplicon sequence data of soil, rhizosphere, and root endosphere microbiomes. This information can be used to investigate the composition and diversity of plant-associated microbial communities, and is suitable for the use with a wide range of plant species.

Esplorare il microbioma radice: estrazione dei dati di comunità batteriche dal suolo e la rizosfera radice Endosphere

The intimate interaction between plant host and associated microorganisms is crucial in determining plant fitness, and can foster improved tolerance to ...

L'uso del Mouse Splenocytes per valutare Pathogen-associated Molecular Pattern influenza sull'espressione del Gene dell'orologio

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Misura parallela di circadiano Clock espressione genica e la secrezione dell'ormone in colture cellulari primarie umane