Name: simultaneous quantification of t-cell receptor excision circles (trecs) and k-deleting recombination excision circles (krecs) by real-time pcr
Uploaded: 2014-12-06T14:00:00
Description: Watch this Scientific Journal Video about Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR at JoVE.com

The authors have no acknowledgements.

OPMERKING: Ethiek verklaring: Dit protocol volgt de richtlijnen van onze instelling, de Spedali Civili di Brescia 1. Voorbereiding van een &quot;Triple-Insert&quot; Plasmid <ol><li> Selectie en de opstelling van een passende uitgangsmateriaal: <ol><li> Het verkrijgen van een monster dat cellen zeer waarschijnlijk TRECS en KRECs detecteerbaar met PCR, zoals perifere bloed van een jonge gezonde onderwerp in EDTA-buizen. NB: Oorspronkelijk hadden we de methode met behulp van thymocytes...

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TREC and KREC quantification can be considered a good estimate of recent thymic and bone marrow output provided that some caveats are taken into account. Even though an absolute quantification method employing standard curve requires more reagents and more space on the real-time PCR reaction plate, it ensures highly accurate quantitative results because unknown sample quantities are interpolated from standard curves built upon known amounts of starting material. Moreover this method is better fitted to detect low amount ...

T-cel receptor excisie cirkels (TRECS) en K verwijderen recombinatie excisie cirkels (KRECs) zijn kleine gecirculariseerde DNA-elementen die zijn uitgesneden in een verhouding van T- en B-cellen, respectievelijk, in een genomisch DNA recombinatie proces leidt tot de vorming van een zeer gediversifieerde repertoire van T- en B-celreceptoren. Zij hebben geen functie, maar omdat ze stabiel zijn en niet kunnen worden gerepliceerd, worden ze verdund na elke celdeling, waardoor aanhoudende slechts één van de twee dochtercellen. Daardoor kan het niveau in het perifere bloed worden aangenomen als een schatting van de thymus en beenmerg output. Terwijl de TREC-test is grotendeels gebruikt in de afgelopen 15 jaar in de mate van thymus uitgang te evalueren, 1 de Krec assay, die aanvankelijk werd ontwikkeld om B-cel proliferatie en de bijdrage ervan aan de B-cel homeostase in gezondheid en ziekte, 2 meten is pas recent voorgesteld als een marker van bot marrow uitgang. 3,4 Hier beschrijven we de methode die we ontwikkeld voor de gelijktijdige kwantificering van zowel TRECS en KRECs. 4 Met deze gecombineerde methode wordt de variabiliteit geassocieerd DNA kwantificatie door real-time PCR geëlimineerd door het gebruik van een unieke standaardcurve verkregen door verdunning van een triple-insertie plasmide bevattende fragmenten van TRECS, KRECs en T-celreceptor alfa constante (TCRAC) gen in een 1: 1: 1 verhouding. Dit maakt een meer nauwkeurige evaluatie van de TREC en Krec copy number. Verder is de gelijktijdige kwantificering van de twee doelen in dezelfde reactie laat reagens kostenreductie. De voorgestelde TREC / Krec test kan nuttig zijn om de omvang van T- en B-cellen neo-productie bij kinderen of volwassenen met ernstige gecombineerde immuundeficiëntie (SCID), 4 Hypogammaglobulinemie, 5 autoimmuunziekten, 6-8 en HIV-infectie meten Voorts. 9, kan het worden gebruikt ombewaken van het immuunsysteem herstel na hematopoietische stamceltransplantatie, 10 enzymvervanging, 11 en antivirale 9 of immunomodulerende therapieën. 6-8 slot omdat SCID patiënten worden opgenomen op basis van TREC test ondanks de onderliggende genetische defecten, en agammaglobulinemie patiënten kunnen worden geïdentificeerd met behulp van Krec kwantificering De TREC / Krec test kan ook worden gebruikt om immuundeficiënties detecteren bij pasgeboren screeningprogramma&#39;s. 12 In dit geval moet de test worden uitgevoerd op DNA geëxtraheerd uit kleine bloedvlekken geblot en gedroogd op filterpapier, moeten zeer gevoelig en specifiek zijn voor deze ziekten, evenals zeer reproduceerbare en kosteneffectief. De invoering van Krec kwantificering in de test prestaties van pasgeboren screening voor immuundeficiënties die routinematig werd uitgevoerd in sommige delen van de Verenigde Staten (WI, MA, CA) sinds 2008 verbeteren wanneer Wisconsin werd eerste Inleidinge de analyse van TRECS in haar postnatale screening programma. 13

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			<td height="21" style="height:21px;width:296px;">Name of Reagent/ Equipment</td>
			<td style="width:211px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:235px;">Comments/Description</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Histopaque-1077</td>
			<td style="width:211px;">Sigma Aldrich SRL</td>
			<td style="width:119px;">10771-500 ML</td>
			<td style="width:235px;">density gradient separation method</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QIAamp DNA Blood Mini Kit (250)</td>
			<td style="width:211px;">QIAGEN</td>
			<td style="width:119px;">51106</td>
			<td style="width:235px;">DNA extraction</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;">Unmodified DNA Oligonucleotides HPSF 0.01 mmol</td>
			<td style="width:211px;">Eurofins MWG Operon/Carlo Erba Reagents S.r.l&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:235px;">Resuspend the lyophilized product to 100 picmol/&#181;l</td>
		</tr>
		<tr height="45">
			<td height="45" style="height:45px;width:296px;">AmpliTaq DNA Polymerase: including 10x Buffer II and 25mM MgCl2</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;">N8080156</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">GeneAmp dNTP Blend (100 mM)</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;">N8080261</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TOPO TA Cloning Kit for Subcloning</td>
			<td style="width:211px;">Invitrogen/Life-Technologies</td>
			<td>K4500-01</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">XL1-Blue Subcloning Grade Competent Cells</td>
			<td style="width:211px;">Stratagene</td>
			<td>200130</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureYield Plasmid Miniprep System</td>
			<td style="width:211px;">Promega</td>
			<td>A1223</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">SpeI 500U</td>
			<td style="width:211px;">New England Biolabs</td>
			<td>R0133S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HindIII-HF 10,000 U</td>
			<td style="width:211px;">New England Biolabs</td>
			<td>R3104S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">PureYield Plasmid Midiprep System</td>
			<td style="width:211px;">Promega</td>
			<td>A2492</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:296px;">XhoI 5,000 U</td>
			<td style="width:211px;">New England Biolabs</td>
			<td>R0146S</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TRIS Utrapure</td>
			<td style="width:211px;">Sigma Aldrich SRL</td>
			<td>T1503</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">EDTA</td>
			<td style="width:211px;">Sigma Aldrich SRL</td>
			<td>E5134</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TE buffer (1 mM TRIS and 0.1 mM EDTA)</td>
			<td style="width:211px;"></td>
			<td style="width:119px;"></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">TaqMan Universal PCR Master Mix</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;">4364338</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dual labeled probes HPLC 0.01 mmol</td>
			<td style="width:211px;">Eurofins MWG Operon/Carlo Erba Reagents S.r.l&#160;</td>
			<td style="width:119px;"></td>
			<td style="width:235px;">Resuspend the lyophilized product to 100 picmol/&#181;l</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">NanoDrop 2000c spectrophotometer</td>
			<td style="width:211px;">ThermoFisher</td>
			<td></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Applied Biosystems 2720 Thermal Cycler</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td>4359659</td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Fast 7500 Real-Time PCR system</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:235px;"></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:296px;">SDS Sequence Detection Software 1.4</td>
			<td style="width:211px;">Applied Biosystems/Life-Technologies</td>
			<td style="width:119px;"></td>
			<td style="width:235px;"></td>
		</tr>
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simultaneous quantification of t-cell receptor excision circles (trecs) and k-deleting recombination excision circles (krecs) by real-time pcr

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination process that creates T- and B-cell receptors. Because TRECs and KRECs are unable to replicate, they are diluted after each cell division, and therefore persist in the cell. Their quantity in peripheral blood can be considered as an estimation of thymic and bone marrow output. By combining well established and commonly used TREC assay with a modified version of KREC assay, we have developed a duplex quantitative real-time PCR that allows quantification of both newly-produced T and B lymphocytes in a single assay. The number of TRECs and KRECs are obtained using a standard curve prepared by serially diluting TREC and KREC signal joints cloned in a bacterial plasmid, together with a fragment of T-cell receptor alpha constant gene that serves as reference gene. Results are reported as number of TRECs and KRECs/106 cells or per ml of blood. The quantification of these DNA fragments have been proven useful for monitoring immune reconstitution following bone marrow transplantation in both children and adults, for improved characterization of immune deficiencies, or for better understanding of certain immunomodulating drug activity.

De test werd uitgevoerd in een representatieve steekproef van 87 gezonde controles: 42 kinderen in de leeftijd 0-17 (man / vrouwtjes: 25/17) en 45 volwassenen in de leeftijd 24-60 (mannen / vrouwen: 29/16). De resultaten werden verkregen als TRECS en KRECs per 10 6 PBMC, en vervolgens de TRECS en KRECs per ml bloed werden berekend. Het aantal TRECS afneemt met de leeftijd toe te schrijven aan involutie van de thymus, 4 in het bijzonder in een zeer scherpe fashion 0-3 - ...

Watch this Scientific Journal Video about Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR at JoVE.com

Simultaneous Quantification of T-Cell Receptor Excision Circles TRECs and K-Deleting Recombination Excision Circles KRECs by Real-time PCR

Gelijktijdige Kwantificering van de T-cel receptor Excisie cirkels (TRECS) en K-verwijderen recombinatie Excisie cirkels (KRECs) van Real-time PCR

Here, we describe a method for simultaneous quantification of T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs). The TREC/KREC assay can be used as marker of thymic and bone marrow output.

T-cell receptor excision circles (TRECs) and K-deleting recombination excision circles (KRECs) are circularized DNA elements formed during recombination ...

Research

JoVE Journal

Immunology and Infection

Simultaneous Quantification of T-Cell Receptor Excision Circles (TRECs) and K-Deleting Recombination Excision Circles (KRECs) by Real-time PCR

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

Transnuclear Mice with Pre-defined T Cell Receptor Specificities Against Toxoplasma gondii Obtained Via SCNT

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Lymphocytes, such as T cells, undergo genetic V(D)J recombination, to generate a receptor with a certain specificity1. Mice transgenic for a rearranged ...

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Protection against infectious diseases is mediated by the immune system 1,2. T lymphocytes are the master coordinators of the immune system, regulating ...

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Myeloid cells are the most abundant immune cells within tumors and have been shown to promote tumor progression. Modern intravital imaging techniques ...

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High-throughput Kwantitatieve real-time RT-PCR assay voor het bepalen van expressieprofielen van de typen I en III interferon subtypen

Described in this report is a qRT-PCR assay for the analysis of seventeen human IFN subtypes in a 384-well plate format that incorporates highly specific ...

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T cell receptor (TCR) signaling is essential in the development and differentiation of T cells in the thymus and periphery, respectively.  The vast array ...

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genotypering van<em&gt; Staphylococcus aureus</em&gt; Door Ribosomaal Spacer PCR (RS-PCR)

The ribosomal spacer PCR (RS-PCR) is a highly resolving and robust genotyping method for S. aureus that allows a high throughput at moderate costs and is, ...

Real-time Imaging and Quantification of Fungal Biofilm Development Using a Two-Phase Recirculating Flow System

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In oropharyngeal candidiasis, members of the genus Candida must adhere to and grow on the oral mucosal surface while under the effects of salivary flow. ...

Identification of Mediators of T-cell Receptor Signaling via the Screening of Chemical Inhibitor Libraries

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Identificatie van bemiddelaars/Mediators van T-cel Receptor signalering via de Screening van chemische remmer bibliotheken

The T-cell receptor (TCR) signaling pathway comprises a multitude of mediators that transmit signals upon the activation of the TCR. Different strategies ...