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.

Declaração de Ética: NOTA: Este protocolo segue as diretrizes da nossa instituição, o Spedali Civili di Brescia 1. Elaboração de um &quot;Triple-Insert&quot; plasmídeo <ol><li> Selecção e preparação de material de partida apropriado: <ol><li> Obter uma amostra contendo células muito susceptíveis de ter TRECs e KRECs detectável por PCR, tal como sangue periférico de um sujeito jovem saudável recolhido em tubos contendo EDTA. NOTA: Originalmente, tínhamos desenvolvid...

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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 ...

Círculos excisão do receptor de células T (TRECs) e os círculos a recombinação de excisão-exclusão K (KRECs) são pequenos elementos de ADN circularizado que são excisados, numa proporção de células T e B-, respectivamente, durante um processo de recombinação de DNA genómico, levando à formação de um repertório altamente diversificada de T e receptores de células-B. Eles não têm qualquer função, mas porque eles são estáveis ​​e não pode ser replicado, eles são diluídos após cada divisão celular, persistindo assim em apenas uma das duas células filhas. Portanto, os seus níveis no sangue periférico pode ser assumida como uma estimativa da potência do timo e da medula óssea. Enquanto o ensaio de TREC tem sido largamente utilizado nos últimos 15 anos, para avaliar a extensão da saída do timo, a um ensaio de KREC, que foi inicialmente desenvolvido para medir a proliferação de células B e a sua contribuição para a homeostase da célula B na saúde e na doença, 2 Só recentemente foi proposta como um marcador de osso marrow saída 3,4. Aqui, descrevemos o método que desenvolvemos para a quantificação simultânea de ambos os TRECs e KRECs. 4 Com este método combinado, a variabilidade associada a quantificação de ADN por PCR em tempo real é eliminada através da utilização de uma única curva padrão obtida por diluição de um plasmídeo de inserção de triplo contendo fragmentos de TRECs, KRECs e de células T constante receptor alfa (TCRAC) gene numa proporção de 1: 1: 1. Isto permite uma avaliação mais precisa do número de exemplares TREC e KREC. Além disso, a quantificação simultânea dos dois alvos na mesma reacção permite a redução de custos de reagentes. O ensaio de TREC / KREC proposto pode ser útil para medir o grau de T e neo-produção de células B em crianças ou adultos com Imunodeficiência Combinada Grave (SCID), 4 imunodeficiência comum variável, cinco doenças auto-imunes, 6-8 e infecção pelo HIV 9. Além disso, ele pode ser usado paramonitorar a recuperação imunológica após o transplante de células-tronco hematopoéticas, 10 de substituição enzimática, 11 e antiviral 9 ou imunomoduladores terapias. 6-8 Finalmente, porque os pacientes SCID são reconhecidos utilizando o ensaio de TREC apesar dos defeitos genéticos subjacentes, e agammaglobulinemia pacientes podem ser identificados usando KREC quantificação , o ensaio TREC / KREC pode também ser usado para detectar imunodeficiências em programas de rastreio neonatal. 12 Neste caso, o ensaio deve ser realizado em ADN extraído de pequenas manchas de sangue apagados e secos em papel de filtro, deve ser altamente sensível e específico para as doenças-alvo, bem como altamente reprodutível e de baixo custo. A introdução de KREC quantificação do ensaio deve melhorar performances de triagem neonatal para imunodeficiências, que tem sido realizada rotineiramente nos algumas partes dos EUA (WI, MA, CA) desde 2008, quando se tornou o primeiro Wisconsin para Introduce da análise de TRECs em seu programa de triagem pós-natal. 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>
	</tbody>
</table>

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.

O ensaio foi realizado em uma amostra representativa de 87 controles saudáveis: 42 crianças com idades entre 0-17 (masculino / feminino: 25/17) e 45 adultos com idades entre 24-60 (homens / mulheres: 29/16). Os resultados foram obtidos como TRECs e KRECs por 10 6 PBMC, e, em seguida, os TRECs e KRECs por ml de sangue foram calculados. O número de TRECs diminui com a idade, devido a involução do timo, 4, em especial de uma forma muito acentuada de 0 a 3 - 4 anos. Em...

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

Quantificação simultânea de T-célula receptora Excisão Circles (TRECs) e K-Excluindo Recombinação Excisão Circles (KRECs) por PCR em tempo real

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 ...

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

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