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.

OBS: Etik uttalande: Detta protokoll följer riktlinjerna i vår institution, Spedali Ċivili di Brescia 1. Beredning av en &quot;Triple-Insert&quot; plasmid <ol><li> Urval och beredning av lämpligt utgångsmaterial: <ol><li> Skaffa ett prov innehållande celler mycket som kan få TRECs och KRECs detekterbara med PCR, såsom perifert blod av en ung frisk person samlas in EDTA-rör. OBS: Ursprungligen hade vi utvecklat metoden använder tymocyter för TRECs och mononukleära celler ...

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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-cellreceptor excision cirklar (TRECs) och K-strykning rekombination excision cirklar (KRECs) är små cirkulariserade DNA-element som är utskurna i en proportion av T- och B-celler, respektive, under ett genomiskt DNA-rekombination, som kan leda till bildning av en mycket diversifierad repertoar av T- och B-cellreceptorer. De har ingen funktion, utan för att de är stabila och inte kan replik, de späds efter varje celldelning, vilket kvarstår endast i en av de två dotterceller. Därför kan deras nivåer i perifert blod antas som en uppskattning av tymus och benmärg utgång. Medan TREC-analysen har till stor del använts under de senaste 15 åren för att bedöma omfattningen av tymus utgång, till 1 av KREC analysen, som ursprungligen utvecklades mäta B-celltillväxt och dess bidrag till B-cells homeostas vid hälsa och sjukdom, 2 har först nyligen föreslagits som en markör för ben marrow utgång. 3,4 Här beskriver vi den metod vi utvecklat för samtidig kvantifiering av både TRECs och KRECs. 4 Med denna kombinerade metod är variabiliteten associerad med DNA-kvantifiering genom realtids-PCR elimineras genom användning av ett unikt standardkurva erhållen genom att späda en trippel-insert plasmid innehållande fragment av TRECs, KRECs och T-cellreceptor-alfa konstant (TCRAC) gen i en 1: 1: 1-förhållande. Detta möjliggör en mer korrekt bedömning av TREC och KREC antal kopior. Vidare, den samtidiga kvantifieringen av de två målen i den samma reaktionen tillåter minskning reagenskostnaden. Den föreslagna TREC / KREC analysen kan vara användbart för att mäta omfattningen av T- och B-cells neo-produktionen hos barn eller vuxna med svår kombinerad immunbrist (SCID), 4 vanlig variabel immun, 5 autoimmuna sjukdomar, 6-8 och HIV-infektion . 9 Dessutom kan den användas för attövervaka immun återhämtning efter hematopoetisk stamcellstransplantation, 10 enzymersättnings, 11 och antivirala 9 eller immunomodulerande terapier. 6-8 Slutligen därför SCID patienter redovisas enligt TREC analys trots de underliggande genetiska defekter, och agammaglobulinemi patienter kan identifieras med hjälp KREC kvantifiering , den TREC / KREC analysen kan också användas för att upptäcka immun hos nyfödda screeningprogram. 12 I detta fall skall provningen utföras på DNA extraherat från små fläckar av blod blottades och torkades på filterpapper, måste vara mycket känslig och specifik för målsjukdomarna samt mycket reproducerbar och kostnadseffektivt. Införandet av KREC kvantifiering i testet bör förbättra föreställningar av nyfödda screening för immun, som rutinmässigt har utförts i vissa delar av USA (WI, MA, CA) sedan 2008 då Wisconsin blev den första att inföe analysen av TRECs i sitt postnatal screeningprogram. 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.

Analysen genomfördes i ett representativt urval av 87 friska kontroller: 42 barn i åldern 0-17 (manliga / honor: 25/17) och 45 vuxna i åldern 24-60 (män / kvinnor: 29/16). Resultaten erhölls som TRECs och KRECs per 10 6 PBMC, och sedan de TRECs och KRECs per ml blod beräknades. Antalet TRECs minskar med åldern på grund av tymisk involution, 4 särskilt i en mycket skarp mode 0-3 - 4 år. Hos vuxna, det TREC antalet beror också på kön eftersom det minskar snab...

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

Samtidig Kvantifiering av T-cellreceptor Excision cirklar (TRECs) och K-Radering Rekombination Excision cirklar (KRECs) genom realtids-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

Transnuclear Möss med fördefinierade Receptor T cellspecifika Against Toxoplasma gondii Erhållits via SCNT

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

A TIRF Microscopy Technique for Real-time, Simultaneous Imaging of the TCR and its Associated Signaling Proteins

En TIRF Mikroskopi Teknik för Real-time, samtidig avbildning av TCR och dess associerade signalproteiner

Signaling is initiated through the T Cell Receptor (TCR) when it is engaged by antigenic peptide fragments bound by Major Histocompatibility Complex ...

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Detektering av MikroRNA i Mikroglia av Real-time PCR i normala CNS och under Neuroinflammation

Microglia are cells of the myeloid lineage that reside in the central nervous system (CNS)1. These cells play an important role in pathologies of many ...

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Real-time Live avbildning av T-cellsignalering Complex Formation

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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Realtid avbildning av myeloida celler Dynamics i<em&gt; Apc<sup&gt; Min / +</sup</em&gt; Tarmtumörer genom Spinning Disk konfokalmikroskopi

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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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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Retroviral Transduction av benmärgsstamceller för att generera T-cellreceptor Retrogenic Möss

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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genotypning av<em&gt; Staphylococcus aureus</em&gt; vid Ribosomal 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

Realtid avbildning och kvantifiering av svamp Biofilm utveckling med hjälp av en tvåfas recirkulerande flödessystem

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

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

Identifiering av mediatorer av T-cells Receptor signalering via Screening av kemiska hämmare bibliotek

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