QuantiTect RT-PCR Kits

Para la qRT-PCR de un paso mediante SYBR® Green I y qRT-PCR de un paso y qRT-PCR múltiple mediante sondas específicas de secuencia para el análisis de la expresión génica

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QuantiTect Probe RT-PCR Kit (200)

Cat. No. / ID: 204443

Para 200 reacciones de 50 µl: 3 unidades de 2x QuantiTect Probe RT-PCR Master Mix 1,7 ml, QuantiTect RT Mix 100 µl, 2 unidades de RNase-Free Water 2 ml

Detection type

Probe

SYBR Green

Multiplex Probe

Reactions

200

1000

Inquire

QuantiTect RT-PCR Kits están concebidos para su uso en aplicaciones de biología molecular. Estos productos no están concebidos para el diagnóstico, la prevención ni el tratamiento de enfermedades.

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Características

Detección de dianas con pocas copias muy sensible
qRT-PCR mediante sondas específicas de secuencia o SYBR Green
Cuantificación exacta sobre varios logaritmos del molde
Sin necesidad de optimizar la reacción y las condiciones de termociclado
Detección del gen de referencia y de hasta 3 dianas en el mismo tubo

Detalles del producto

Los QuantiTect RT-PCR Kits permiten la cuantificación sensible de las dianas de ARN mediante la real-time PCR de un paso utilizando sondas específicas de secuencia o detección SYBR Green I. Los kits también permiten la cuantificación fiable de hasta 5 dianas de ARN en un único tubo mediante la RT-PCR de un paso en tiempo real y múltiple. La combinación de un hot start y un sistema de tampón de PCR exclusivo en la mezcla maestra lista para usar garantiza una qRT-PCR de alta sensibilidad en cualquier termociclador en tiempo real sin necesidad de optimización. La mezcla dNTP incluye dUTP, lo que permite el tratamiento opcional con UNG. Por motivos de conveniencia, las mezclas maestras en los kits de RT-PCR de un paso de QuantiTect pueden almacenarse a una temperatura de 2-8 °C.

Hay dos formatos de kits disponibles para la RT-PCR múltiple utilizando sondas específicas de secuencia: el QuantiTect Multiplex RT-PCR Kit para termocicladores que necesitan un colorante ROX para la normalización de la fluorescencia y el QuantiTect Multiplex RT-PCR NoROX Kit para el resto de termocicladores. El QuantiTect SYBR Green RT-PCR Kit se suministra con una mezcla de RT optimizada para una transcripción inversa sensible y eficaz en una gran variedad de cantidades de moldes de ARN.

Rendimiento

La HotStarTaq DNA Polymerase incluida en los kits de RT-PCR de un paso de QuantiTect aumenta la especificidad de la reacción de PCR al proporcionar el hot start más estricto en comparación con otras polimerasas.
El QuantiTect SYBR Green RT-PCR Kit contiene una mezcla única de transcriptasas inversas que proporciona una síntesis de ADNc sensible y eficaz en una gran variedad de cantidades de moldes de ARN y permite una cuantificación específica en un amplio intervalo lineal (consulte la figura “ Resultados comparables en la RT-PCR de un paso y de dos pasos”).

El QuantiTect Probe RT-PCR Kit contiene un tampón de RT-PCR exclusivo que favorece la hibridación altamente específica de sondas y cebadores al molde de PCR. La HotStarTaq DNA Polymerase y la composición exclusiva del tampón de RT-PCR permiten que el QuantiTect Probe RT-PCR Kit proporcione una cuantificación sensible de dianas de ARN de pocas copias, así como una cuantificación exacta en un amplio intervalo lineal (consulte la figura “ Alta sensibilidad y eficacia y amplio rango dinámico”).

Llevar a cabo la transcripción inversa y la PCR secuencialmente en el mismo tubo no merma la sensibilidad, tal y como demuestran los valores de CT comparables a aquellos obtenidos en la RT-PCR en tiempo real de dos pasos (consulte las figuras “ RT-PCR de un paso con rendimiento comparable a la RT-PCR de dos pasos: A” y “ RT-PCR de un paso con rendimiento comparable a la RT-PCR de dos pasos: B”, y la tabla “RT-PCR de un paso con rendimiento comparable a la RT-PCR de dos pasos”).

RT-PCR de un paso con rendimiento comparable a la RT-PCR de dos pasos.

Cantidad de ADNc/ARN	Valor de C_T medio (RT-PCR de dos pasos)	Valor de C_T medio (RT-PCR de un paso)
100 ng	24,66	24,66
10 ng	28,15	27,84
1 ng	31,42	31,49
0,1 ng	34,80	34,56
0,01 ng	37,78	37,66

La detección sensible de tan solo 10 copias de la secuencia diana (consulte la figura “ Detección de hasta 10 copias del ARN diana en PCR doble”) puede lograrse en la qRT-PCR múltiple con los QuantiTect Multiplex RT-PCR Kits. La cuantificación relativa precisa de la expresión de un gen se consigue a través de la cuantificación de la expresión tanto del gen diana como de un gen de control endógeno en el mismo pocillo o tubo. Con los QuantiTect Multiplex RT-PCR Kits, los valores de CT de las dianas en una reacción múltiple son equivalentes a aquellos obtenidos en los experimentos de control, donde las dianas se amplifican en reacciones separadas (consulte la figura “ Amplificación comparable en la PCR triple y las PCR simples”). Esto demuestra que las dianas diferentes en la misma reacción múltiple se amplifican de forma eficaz y sensible sin influirse entre ellas.

Ver figuras

Resultados comparables en la RT-PCR de un paso y de dos pasos.

Alta sensibilidad y eficacia y amplio rango dinámico.

RT-PCR de un paso con rendimiento comparable a la RT-PCR de dos pasos: A.

RT-PCR de un paso con rendimiento comparable a la RT-PCR de dos pasos: B.

Detección de hasta 10 copias del ARN diana en PCR doble.

Amplificación comparable en la PCR triple y las PCR simples.

Principio

Los QuantiTect RT-PCR Kits contienen una mezcla maestra optimizada y lista para usar para una cuantificación en tiempo real muy específica y sensible de dianas de ARN utilizando SYBR Green I o sondas específicas de secuencia. El colorante fluorescente SYBR Green I en la mezcla maestra de QuantiTect SYBR Green RT-PCR permite el análisis de muchas dianas diferentes sin necesidad de sintetizar sondas marcadas como específicas de la diana (consulte la tabla “Componentes del 2x QuantiTect SYBR Green RT-PCR Kit”).

Componentes del 2x QuantiTect SYBR Green RT-PCR Kit

Componente	Características	Beneficios
HotStarTaq DNA Polymerase	Activación de 15 min a 95 ºC	Preparación de reacciones de qPCR a temperatura ambiente
QuantiTect SYBR Green RT-PCR Buffer	Combinación equilibrada de iones de NH₄⁺ y K⁺	La hibridación de cebadores específicos garantiza la fiabilidad de los resultados de la PCR
Mezcla dNTP	Incluye dUTP, que sustituye parcialmente al dTTP y permite el tratamiento opcional de las reacciones con UNG	Elimina la contaminación causada por el arrastre de productos de PCR mediante el tratamiento opcional con UNG
Colorante SYBR Green I	Produce una señal fluorescente intensa al unirse al ADN bicatenario	Cuantificación muy sensible
Colorante ROX	Para la normalización de las señales fluorescentes en instrumentos de Applied Biosystems y, opcionalmente, de Agilent	Cuantificación precisa en termocicladores que requieren colorante ROX. No interfiere en las reacciones en otros termocicladores en tiempo real
Omniscript y Sensiscript Reverse Transcriptases (transcriptasas inversas Omniscript y Sensiscript)	Mezcla especial de enzimas con alta afinidad por el ARN	El ARN puede transcribirse, incluso a través de estructuras secundarias complejas

Los QuantiTect Probe RT-PCR Kits contienen una mezcla maestra optimizada y lista para usar para la cuantificación en tiempo real muy específica y sensible de dianas de ARN utilizando sondas específicas de secuencia (consulte la tabla “Componentes del 2x QuantiTect Probe RT-PCR Kit”). Los kits están diseñados para usarse con todos los tipos de sondas específicas de secuencia, incluidas las sondas de hidrólisis (p. ej., TaqMan® y otras sondas con doble marcado), sondas FRET y balizas moleculares.

Componentes del 2x QuantiTect Probe RT-PCR Kit

Componente	Características	Beneficios
HotStarTaq DNA Polymerase	Activación de 15 min a 95 ºC	Preparación de reacciones de qPCR a temperatura ambiente
QuantiTect Probe RT-PCR Buffer	Combinación equilibrada de iones de NH₄⁺ y K⁺	La hibridación de cebadores específicos garantiza la fiabilidad de los resultados de la PCR
Mezcla dNTP	Incluye dUTP, que sustituye parcialmente al dTTP y permite el tratamiento opcional de las reacciones con UNG	Elimina la contaminación causada por el arrastre de productos de PCR mediante el tratamiento opcional con UNG
Colorante SYBR Green I	Produce una señal fluorescente intensa al unirse al ADN bicatenario	Cuantificación muy sensible
Colorante ROX	Para la normalización de las señales fluorescentes en instrumentos de Applied Biosystems y, opcionalmente, de Agilent	Cuantificación precisa en termocicladores que requieren colorante ROX. No interfiere en las reacciones en otros termocicladores en tiempo real
Omniscript y Sensiscript Reverse Transcriptases (transcriptasas inversas Omniscript y Sensiscript)	Mezcla especial de enzimas con alta afinidad por el ARN	El ARN puede transcribirse, incluso a través de estructuras secundarias complejas

La cuantificación relativa precisa de la expresión de un gen se consigue a través de la cuantificación de la expresión tanto del gen diana como de un gen de control endógeno en el mismo pocillo o tubo. La mezcla maestra optimizada en el QuantiTect Multiplex RT-PCR Kit garantiza que los productos de PCR de una reacción múltiple se amplifiquen con la misma eficacia y sensibilidad que los productos de PCR en la reacción de amplificación simple correspondiente (consulte la tabla “Componentes del 2x QuantiTect Multiplex RT-PCR Kit”). Con el kit se pueden detectar tan solo 10 copias de una diana.

Componentes del 2x QuantiTect Multiplex RT-PCR Kit

Componente	Características	Beneficios
HotStarTaq DNA Polymerase	Activación de 15 min a 95 ºC	Preparación de reacciones de qPCR a temperatura ambiente
QuantiTect Multiplex RT-PCR Buffer	Combinación equilibrada de iones de NH₄⁺ y K⁺	La hibridación de cebadores específicos garantiza la fiabilidad de los resultados de la PCR
QuantiTect Multiplex RT-PCR Buffer	Factor MP sintético	Análisis múltiple fiable de hasta 4 genes en el mismo tubo
Mezcla dNTP	Incluye dUTP, que sustituye parcialmente al dTTP y permite el tratamiento opcional de las reacciones con UNG	Elimina la contaminación causada por el arrastre de productos de PCR mediante el tratamiento opcional con UNG
Colorante ROX*	Para la normalización de las señales fluorescentes en instrumentos de Applied Biosystems y, opcionalmente, de Agilent	Cuantificación precisa en termocicladores que requieren colorante ROX. No interfiere en las reacciones en otros termocicladores en tiempo real
Omniscript y Sensiscript Reverse Transcriptases (transcriptasas inversas Omniscript y Sensiscript)	Mezcla especial de enzimas con alta afinidad por el ARN	El ARN puede transcribirse, incluso a través de estructuras secundarias complejas

La combinación equilibrada de iones de K⁺ y NH₄⁺ en el tampón de PCR de los QuantiTect RT-PCR Kits –junto con el Factor MP sintético del tampón de RT-PCR QuantiTect Multiplex– favorece la hibridación de cebadores específicos y permite un alto nivel de especificidad y sensibilidad en la RT-PCR (consulte la figura “ Hibridación de cebadores específicos”). Además, una mezcla optimizada de transcriptasas inversas permite la síntesis de ADNc de una gran variedad de cantidades de moldes de ARN, mientras que la HotStarTaq DNA Polymerase proporciona un riguroso hot start, lo que impide la formación de productos no específicos.

Las mezclas maestras del QuantiTect RT-PCR también contienen dUTP, lo que permite el pretratamiento con uracilo N-glicosilasa (UNG) antes de iniciar la PCR, que garantiza que las reacciones de PCR posteriores no se vean afectadas por productos de PCR contaminantes.

Ver figuras

Hibridación de cebadores específicos.

Procedimiento

Los QuantiTect RT-PCR Kits no hacen necesaria la optimización de las condiciones de reacción, lo que puede ser tedioso y demandar mucho tiempo. Simplemente, añada los cebadores y el molde a la mezcla maestra lista para usar del QuantiTect SYBR Green RT-PCR –o los cebadores, la sonda y el molde a la mezcla maestra lista para usar del QuantiTect Probe RT-PCR– e inicie la reacción (consulte el organigrama “ RT-PCR de un paso con SYBR Green” y “ RT-PCR de un paso con sondas específicas de secuencia”). Siga el protocolo del manual de uso para obtener resultados rápidos y fiables en cualquier termociclador en tiempo real. Si es necesario, se puede realizar el pretratamiento de las reacciones con uracilo N-glicosilasa (UNG) para eliminar el arrastre de productos de PCR de otras reacciones.

Se garantizan unos resultados muy específicos en el análisis de expresión génica cuando se utilizan los QuantiTect SYBR Green RT-PCR Kits en combinación con los QuantiTect Primer Assays. Se trata de conjuntos de cebadores de genoma completo validados por vía bioinformática destinados a la detección de transcritos de muestras humanas, de ratón, rata y muchas otras. Los QuantiTect Primer Assays se pueden pedir fácilmente en línea en GeneGlobe.

El manual de uso de los QuantiTect Multiplex RT-PCR Kits contiene un protocolo único que puede utilizarse con todos los termocicladores en tiempo real disponibles e incorpora una lista de los colorantes recomendados. Los kits están disponibles con colorante de referencia pasivo ROX en la mezcla maestra o sin él, lo que permite su uso en prácticamente cualquier termociclador en tiempo real (consulte la tabla “Elegir el QuantiTect Multiplex RT-PCR Kit correcto”). Gracias a las concentraciones optimizadas de ROX, se logra la detección incluso de números de copias bajos a través del análisis automático de los datos.

Elegir el QuantiTect Multiplex RT-PCR Kit correcto

Colorante ROX	Kit	Termocicladores compatibles
Se suministra en la mezcla maestra	QuantiTect Multiplex RT-PCR Kit	Termocicladores de Applied Biosystems
No se suministra en la mezcla maestra	QuantiTect Multiplex RT-PCR NR Kit	Termocicladores Rotor-Gene y termocicladores de Bio-Rad, Cepheid, Eppendorf, Roche, Agilent y otros proveedores

Ver figuras

RT-PCR de un paso con SYBR Green.

RT-PCR de un paso con sondas específicas de secuencia.

Aplicaciones

Los QuantiTect RT-PCR Kits pueden utilizarse para el análisis de la expresión génica de las dianas de ARN en cualquier termociclador en tiempo real. Esto incluye instrumentos de Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche y Agilent. Para el Rotor-Gene Q y otros termocicladores Rotor-Gene, recomendamos el uso del Rotor-Gene Probe RT-PCR Kit, del Rotor-Gene Multiplex RT-PCR Kit o del Rotor-Gene SYBR Green RT-PCR Kit, que se han desarrollado especialmente para el termociclado rápido en esos instrumentos.

Comparación de QuantiTect RT-PCR Kits
Características	QuantiTect SYBR Green RT-PCR Kit	QuantiTect Probe RT-PCR Kit	QuantiTect Multiplex RT-PCR Kits
Aplicaciones	Cuantificación de dianas de ARN en tiempo real	Cuantificación de dianas de ARN en tiempo real	Cuantificación de dianas de ARN en tiempo real en un formato múltiple
Tipo de reacción	RT-PCR de un paso	RT-PCR de un paso	RT-PCR de un paso múltiple
En tiempo real o punto final	En tiempo real	En tiempo real	En tiempo real
Tipo de muestra/diana	ARN	ARN	ARN
Único o múltiple	Único	Único	Único
SYBR Green I o sondas específicas de secuencia	SYBR Green I	Sondas específicas de secuencia	Sondas específicas de secuencia
Termociclador	Todos los termocicladores en tiempo real (p. ej., LightCycler, Rotor-Gene, ABI)	Todos los termocicladores en tiempo real (p. ej., LightCycler, Rotor-Gene, ABI)	Termocicladores en tiempo real dedicados a la PCR múltiple (p. ej., la mayoría de termocicladores en tiempo real de Applied Biosystems, Roche LightCycler 480, y Bio-Rad iCycler iQ)
Con o sin ROX	Con ROX	Con ROX	Con o sin ROX

Datos y cifras de respaldo

Resultados comparables en la RT-PCR de un paso y de dos pasos.

Se analizaron el ARN total (de 10 ng a 100 pg) o las cantidades equivalentes de ADNc de células HeLa mediante el QuantiTect Primer Assay para MAPK14 humano y los kits indicados. Se obtuvieron valores de C_T similares en la RT-PCR de un paso y de dos pasos.

Recursos

For quantitative, real-time one-step RT-PCR using SYBR Green I

For quantitative, real-time one-step RT-PCR using sequence-specific probes

For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection

QuantiTect Multiplex RT-PCR Kit - with master mix containing ROX passive reference dye QuantiTect Multiplex RT-PCR NR Kit - with master mix free of ROX passive reference dye For gene expression analysis by quantitative, multiplex, real-time one-step RT-PCR using sequence-specific probes

FAQ

QuantiTect SYBR Green PCR Kits contain buffers with different salt concentrations compared to Roche Kits, resulting in different template melting temperatures. We recommend to decrease the annealing temperature by at least by 5°C when using a QuaniTect SYBR Green PCR Kit.

FAQ ID -1083

Ensure PCR amplicons are as short as possible, ideally 60–150 bp. Always use the same algorithm or software to design the primers and probes. For optimal results, only combine assays that have been designed using the same parameters.

Check the functionality of each set of primers and probes in individual assays before combining the different sets in the multiplex assay. Choose compatible reporters and quenchers based on a specific instrument. See How do I select appropriate reporter and quencher combinations for multiplex PCR.

FAQ ID -9093

SYBR Green dye does not interfere with ethidium bromide staining, and realtime SYBR Green PCR products can be visualized on an agarose gel.

FAQ ID -312

RNA purified with the QIAamp Viral RNA Mini Kit has been used for quantification by qPCR with QuantiTect Probe RT-PCR Kit on QIAGEN Rotor-Gene Q.

3828

Yes, please visit our website section 'Using endogenous control genes in real-time RT-PCR' for general information. It provides a list of relative gene expression levels for commonly used human and mouse reference genes.

We offer a set of ready-to-order control genes for use in SYBR Green based as well as probe based real-time RT-PCR.

QuantiTect Primer Assays are primer sets for use in SYBR Green based real-time RT-PCR analysis.
QuantiFast Probe Assays are primer-probe sets for use in dual-labeled probe based real-time RT-PCR.

In addition, you may want to refer to the following citations on reference gene selection for quantitative real-time PCR:

• Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, DePaepe A, Speleman F [2002]: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3:0034.

• Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A., 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 313(4): 856-62.

• Katrien Smits,Karen Goossens, Ann Van Soom, Jan Govaere, Maarten Hoogewijs, Emilie Vanhaesebrouck,Cesare Galli, Silvia Colleoni, Jo Vandesompele, and Luc Peelman [2009]Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts. BMC Res Notes. Dec 11;2:246.

FAQ ID -2371

In one-step RT-PCR, both reverse transcription and amplification are performed in the same tube. Upon completion of reverse transcription, the reaction temperature is raised to reach denaturation/PCR enzyme activation temperature and the thermal cycling (PCR) begins. One-step RT-PCR generally uses gene-specific primers for both the RT and PCR steps. The procedure is fast, easy to automate, and minimizes the risk of contamination due to fewer handling steps.

In two-step RT-PCR, the RNA is first transcribed into cDNA using oligo-dT primers, random oligos, or gene-specific primers. An aliquot of the RT reaction is subsequently added to the real-time PCR reaction in a second tube. Choice of different types of RT primers allows the analysis of different transcripts by PCR from one RT reaction. Most commonly, an oligo-dT primer is used for the RT step, followed by PCR with a pair of gene-specific primers. Precious RNA samples can be immediately transcribed into more stable cDNA for later use and long-term storage.

The advantages of each method are summarized below:

Two-step RT-PCR	One-step RT-PCR
Multiple PCRs from one RT reaction	Easy handling
Flexibility with RT primer choice	Fast procedure
Enables long-term storage of cDNA	High reproducibility
	Low contamination risk

Optimized one-step and two-step RT-PCR kits compatible with any real-time cycler are available from QIAGEN. For further details, please see our online section on 'Critical factors for successful gene expression assays', or download our Brochure 'Critical Factors for Successful Real-Time PCR'.

FAQ ID -1056

No. Uracil-N-Glycosylase will remain active during the reverse-transcription step with the QuantiTect Probe RT-PCR Kit, and destroy any newly synthesized cDNA. Heat-labile UNG is required for one-step RT-PCR. See also FAQ 564 for further info.

FAQ ID -2135

No. Due to hardware limitations, the maximum capacity of these real-time cyclers is triplex PCR.

FAQ ID -716

The reduced reaction volume recommended for QuantiFast PCR Kits compared to QuantiTect PCR Kits allows more efficient temperature transfer during short cycling steps.

FAQ ID -1447

The presence of ROX as passive reference dye in the Master Mix of the QuantiTect Multiplex PCR Kit and the QuantiFast Mutliplex PCR Kit limits the use of these kits to triplex PCR on 4-channel real-time cyclers. This is because the ROX dye occupies the channel for detecting probes labeled with ROX, Texas Red, or other equivalent dyes.

However, 4-plex PCR is possible on on instruments equipped with at least 5 channels. Alternatively, if you have a 4-channel real-time cycler that does not use ROX passive reference dye (e.g., iCycler iQ, Rotor-Gene 3000, Mx4000, Mx3000P, Smart Cycler II, LightCycler 2.0), you can use the QuantiTect Multiplex PCR NoROX Kit or the QuantiFast Multiplex PCR +R Kit to perform 4-plex PCR.

Please see table “Real-Time, Multiplex PCR on a Wide Range of Real-Time Cyclers” for compabilities of the QuantiTect Multiplex kits with different real-time cyclers.

FAQ ID -715

Replicates in real-time PCR may have different plateau heights due to differences in the reaction kinetics for each sample. Even though replicates start out with identical template amounts, the rate at which reagents are being depleted, and the point when exponential accumulation of PCR product stops and becomes linear, differ between replicates. This will result in different plateau heights, the stage where PCR reactions have come to a halt, and little or no additional PCR product is being amplified. You can find further information in Chapter 'Quantification of target amounts' of our Brochure "Critical Factors for Successful Real-Time PCR".

FAQ ID -539

Fluorescent oligonucleotides should be stored in the dark, as light can slowly degrade the fluorescent moieties. For optimal long-term storage of fluorescent dye-labeled probes (except Cyanine 570, Cy3.5, Cyanine 670, and Cy5.5), the oligos should be resuspended in a slightly basic solution (e.g., TE buffer at pH 8.0). If resuspended below pH 7.0, the probe can degrade. We recommend to aliquot the sample, and store the aliquots at -20°C.

Note that Cyanine 570, Cy3.5, Cyanine 670, and Cy5.5 begin to degrade at a pH above pH 7.0. For best results, resuspend Cy-labeled oligos at pH 7.0, aliquot, lyophilize, and store at -20°C.

FAQ ID -784

The ice step is recommended to prevent non-specific reverse transcription during reaction set-up. Mispriming by Taq is not a concern in these cases because both QuantiTect RT-PCR and PCR kits contain HotStarTaq, a DNA polymerase that only becomes active following a 15 minute, 95 degree incubation step.

FAQ ID -283

No. The master mixes in Rotor-Gene Kits contain dTTP instead of dUTP. If UNG treatment is required, we recommend using QuantiTect +UNG Kits. QuantiTect Kits are also compatible with the Rotor-Gene Q; however, the kits require a significantly longer cycling time.

FAQ ID -2117

Yes. The pre-optimized master mix ensures that all gene targets in a multiplex reaction are amplified with the same efficiency and sensitivity as in corresponding "single-plex" reactions, independent of starting copy numbers.

FAQ ID -718

Any DNA contamination will artificially inflate the SYBR Green signal, yielding skewed gene expression profiles and false-positive signals. The most common source of DNA contamination is from PCR products generated during previous experiments. Such contamination is most often due to the improper disposal of tubes, tips, and gels that previously came into contact with PCR products. Additionally, PCR products may also contaminate pipettors, racks, work pads, and commonly used reagents such as water and buffers. To minimize the risk of contaminating your experiment with extraneous DNA, the following steps should be taken:

Remove a single aliquot of water from your PCR-grade stock, sufficient to complete the experiment. This minimizes the number of times that the stock container is opened, thereby minimizing contamination risks.
Use only fresh PCR-grade reagents and disposable labware.
Treat any labware (tubes, tips, and tip boxes) used in PCR with 10% bleach, before discarding.
Maintain a dedicated workspace for PCR setup (perhaps a PCR-only hood), away from areas of the lab where post-PCR work is done, such as running gels, enzyme digestions, and cloning.
Change the lab bench pads/papers often and decontaminate lab benches and labware (racks, pipettors, etc.) before each use by washing with 10% bleach, and/or exposing to UV light for at least 10 minutes. This serves to degrade and/or inactivate contaminating DNA.
Before, during, and after the experiment, minimize the opening and closing of any tubes or plates used during the experiment.

FAQ ID -2654

For accurate quantification we recommend an amplicon length of 100-150 bp to ensure optimal amplification efficiency.

FAQ ID -553

QuantiTect Kits for real-time PCR and RT-PCR have been optimized for a final reaction volume of 50 µl for use with single tubes and 96-well plates, or 20 µl for use with 384-well plates and LightCycler capillaries. We strongly recommend using the primer and probe concentrations, reaction volumes, and amount of starting template given in the QuantiTect handbooks. However, volumes may be reduced to 20 µl and 10 µl, respectively.

FAQ ID -551

Recombinant DNA (recDNA) is very stable and represents the average size of mRNA. Due to the cloning and purification processes, obtaining recDNA can lengthen the overall process of generating standards.

Recombinant RNA (recRNA) and native RNA undergo reverse transcription as well as PCR, and mimic the natural process for mRNA in RT-PCR. Complicated cloning and purification of recRNA and instability of recRNA are two disadvantages for using recRNA as a standard. For further details please refer to the section "Generating Standard Curves" in Appendix D of the QuantiTect SYBR Green PCR Handbook.

FAQ ID -729

QuantiTect Primer Assays are primer pairs designed and bioinformatically validated specifically for real-time RT-PCR with SYBR Green detection. To find a primer assay for your target gene of interest, please visit our GeneGlobe data base.

For best results, we strongly recommend using QuantiTect Primer Assays in combination with QIAGEN's products for SYBR Green-based Real-Time PCR and RT-PCR.

FAQ ID -1141

The primers of the QuantiTect Primer Assays are at a proprietary concentration that was specially optimized for sensitive and efficient amplification in any real time cycler. Always dilute these primers to a final work solution of 1x in your reaction, using either the QuantiTect SYBR Green PCR Kit or the QuantiTect SYBR Green RT-PCR Kit. Follow the instructions for use of the Primer Assays in the QuantiTect Primer Assay handbook.

FAQ ID -850

No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.

FAQ ID -9092

No, please do not exchange Quantiscript Reverse Transcriptase of the QuantiTect Reverse Transcription Kit with QuantiTect RT Mix of the QuantiTect Probe RT-PCR Kit.

Although both are an optimized mixture of Omniscript and Sensiscript Reverse Transcriptases, the mixture provided in the QuantiTect Reverse Transription Kit is optimized for random priming in a two-step reaction, whereas the mixture in the QuantiTect Probe RT-PCR Kit is optimized for gene-specific priming in a one-step RT-PCR reaction.

FAQ ID -1066

Yes. The QuantiTect Master Mix may not necessarily freeze at -20 degrees. The performance is not affected as long as the correct storage temperature is maintained.

-20

Quenched FRET assays are similar to FRET assays except that the decrease in energy of the donor fluorophore is measured instead of the increase in energy of the acceptor fluorophore.

FAQ ID -542

The annealing temperature for QuantiTect Primer Assays should be 55^oC when used with the QuantiTect SYBR Green PCR Kit and the QuantiTect SYBR Green RT-PCR Kit.

Use of the QuantiFast SYBR Green PCR and QuantiFast SYBR Green RT-PCR Kits requires a combined annealing/extension step at 60^oC, as described in the QuantiTect Primer Assay Handbook.

Note that these Assays are guaranteed for use with the QuantiTect or QuantiFast chemistries only!

FAQ ID -849

The combination of Omniscript and Sensiscript Reverse Transcriptases was optimized in the QuantiFast RT-PCR Kits. In addition, an optimized dNTP concentration and the limitation of amplicon size to <300 bp allow to reduce the time for the reverse transcription step to only 10 minutes.

FAQ ID -1451

The storage time for QuantiFast PCR Kits is shorter than for QuantiTect PCR Kits, because all QuantiFast master mixes contain HotStarTaq Plus DNA Polymerase, instead of HotStarTaq DNA Polymerase which requires longer activation times.

Excessive exposure to elevated temperatures will result in reactivation of the HotStarTaq Plus DNA Polymerase, eventually leading to nonspecific amplification.

FAQ ID -1446

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

FAQ ID -90990

The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.

FAQ ID -9095

Yes, QuantiTect Primer Assays work very well under fast-cycling conditions. Results achieved with QuantiFast SYBR Green Kits are comparable to those achieved with QuantiTect SYBR Green Kits.

FAQ ID -1714

No, UNG is only available in combination with a QuantiTect PCR Kit.

FAQ ID -2134

The most important prerequisite for any gene expression analysis experiment is the preparation of consistently high-quality RNA from every experimental sample. Contamination by DNA, protein, polysaccharide, or organic solvents can jeopardize the success of an experiment.

Genomic DNA contamination in an RNA sample compromises the quality of gene expression analysis results. The contaminating DNA inflates the OD reading of the RNA concentration. It is also a source of false positive signals in RT-PCR experiments.

RNase contamination degrades RNA samples whichcauses low signal and false-negative results in PCR.

Residual polysaccharides, collagen, other macromolecules, and organic solvents in an RNA sample can inhibit the activity of DNase, which may interfere with DNase treatment for genomic DNA removal. These contaminants may also inhibit reverse transcriptase and DNA polymerase, leading to lower reverse transcription efficiency and reduced PCR sensitivity.

For fast purification of high-quality RNA we recommend QIAGEN’s RNeasy Kits like the RNeasy Mini Kit, the RNeasy Plus Universal Kit, or the RNeasy FFPE Kit.

FAQ ID -2655

The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.

FAQ ID -2682

During the late phase of PCR, as a lot of PCR product has been generated, there is strong competition between hybridization of the probe to the target strand and re-association of the two complementary product strands. For some primer–probe combinations, re-association occurs more quickly than probe hybridization towards the end of the PCR. Therefore, the yield of PCR product seems to decrease after reaching a peak. For accurate quantification, fluorescence data is measured during the log-linear phase of the reaction, i.e., during the initial increase of fluorescence, before this phenomenon occurs.

FAQ ID -587

For each QuantiTect Primer Assay, we retrieve the sequence of the target gene from curated databases and exclude SNP regions from assay design.

We then use our proprietary algorithm to design assays that amplify RNA sequences only (i.e., at least one primer overlaps a splice site), provided that information on the position of splice sites is available. The assays are designed to provide optimal performance with QuantiFast and QuantiTect SYBR Green Kits.

After assay design, we validate randomly selected QuantiTect Primer Assays using real-time RT-PCR to check their compatibility with various real-time cyclers. Both two-step and one-step RT-PCR are carried out. Successful amplification of the target is indicated by the following factors: high sensitivity, high efficiency, high specificity (i.e., a single peak in melting curve analysis), and no primer–dimers in the no-template control (NTC).

To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.

FAQ ID -1982

No, that is not necessary. Simply use the primer concentrations specified in the protocols in the supplied QuantiTect Multiplex PCR handbooks.

FAQ ID -714

Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.

Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line. Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.

If the issue persists, please send the original run file to QIAGEN Technical Services.

FAQ ID -9091

QuantiTect Primer Assays are optimized for an annealing temperature of 55ºC when using the QuantiTect SYBR Green PCR Kit (for two-step RT-PCR) or the QuantiTect SYBR Green RT-PCR Kit (for one-step RT-PCR) .

Increasing the annealing temperature will reduce PCR performance (sensitivity and PCR efficiency) when using QuantiTect Primer Assays with these kits.

FAQ ID -1146

Yes, please follow the Supplementary Protocol 'Performing quantitative, real-time, duplex, triplex, or 4-plex PCR on the LightCycler 2.0 system using the QuantiTect Multiplex PCR NoROX Kit' (PCR44).

FAQ ID -966

For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher^®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.

FAQ ID -9094

Yes, please follow the Supplementary Protocol 'Adding new reporter dyes to the ABI PRISM 7900 Sequence Detection System for use in quantitative, real-time, triplex PCR with the QuantiTect Multiplex PCR Kit' (PCR43).

FAQ ID -965

The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.

FAQ ID -9098

Make sure that template, primers, probes, and amplification reagents are stored correctly and avoid multiple freeze–thaw cycles for oligonucleotides and template. Check the performance of your real-time instrument as some instruments require the halogen lamp to be frequently replaced. Lasers must also be replaced occasionally.

FAQ ID -589

We highly recommend any QuantiTect or QuantiFast Kit for quantitative PCR on cDNA generated with the QuantiTect Whole Transcriptome Kit.

FAQ ID -1592

Depending on the qPCR instrument, time savings when switching from standard cycling (e.g., QuantiTect PCR Kits) to fast cycling using QuantiFast Kits range from 40% to 60%.

FAQ ID -1438

Even when detecting low-abundance targets, we recommend using no more than 500 ng/reaction of DNA (for PCR kits) or RNA (for RT-PCR kits). Generally, 1–100 ng template should be sufficient and for abundant transcripts as little as 1 pg can be used.

Template purity is important if large volumes of low concentration template are to be added to the reaction. Using DNA or RNA purified with QIAGEN products, the template can contribute up to 40% of the final reaction volume as long as the recommended template amounts are not exceeded. If cDNA from an RT reaction is used as template, the volume of undiluted RT reaction added should not exceed 10% of the final PCR volume.

FAQ ID -1086

Store the standards at a high concentration in aliquots at -20^oC to -70^oC. If using low concentrations, stabilize standards with carrier nucleic acid. It is always best to use freshly diluted standards for each experiment. If possible, use siliconized tubes for standard (and target) dilutions. This will prevent any unspecific binding of nucleic acids to the plastic.

FAQ ID -9099

The LightCycler 2.0 requires a color compensation file to separate the fluorescent signals and eliminate crosstalk between the individual detection channels. Please contact QIAGEN Technical Services for a specialized protocol.

FAQ ID -780

Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:

Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
DNA template concentrations should be normalized using the same dilution buffer. Ensure the C_T values are below 30 and differ no more than 3 C_T values across individual samples.
Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
Always start with 0.7 µM primer concentration

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

FAQ ID -9097

No, QuantiTect Primer Assays are supplied as lyophilized, premixed primer pairs. Reaction components for SYBR Green real-time RT-PCR must be purchased separately.

For two-step RT-PCR, the QuantiTect Reverse Transcription Kit and QuantiTect SYBR Green PCR Kit are recommended
For one-step RT-PCR, the QuantiTect SYBR Green RT-PCR Kit is recommended

To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.

FAQ ID -1143

Compatible reporter dye combinations for different real-time cyclers are listed in the "Important Notes" section under 'Suitable combinations of reporter dyes' in the QuantiTect Multiplex PCR Handbook and the QuantiTect Multiplex PCR NoROX Handbook.

FAQ ID -719

No. In fact, QuantiTect SYBR Green PCR Kits contain a stabilization reagent that increases the sensitivity of real-time PCR. The QuantiTect SYBR Green Kits also contain a carefully controlled concentration of SYBR Green that is sufficient to detect PCR fragments while having no inhibitory effect on the enzymatic reaction.

FAQ ID -328

Shorter amplification products facilitate high PCR efficiencies. Ideally, amplicon length should be less than 150 bp for optimal amplification efficiency. PCR efficiencies close to 100% are a crucial prerequisite for accurate quantification of target copy numbers in real-time PCR.

FAQ ID-751

For quantification of RNA, we strongly recommend using RNA molecules as standards. Use of in vitro transcripts as standards takes into account the variable efficiency of the RT reaction. An alternative to the use of in vitro transcripts as RNA standards is the use of a defined RNA preparation (e.g., from a cell line or virus preparation), for which the absolute concentration of the target has already been determined.

For quantification of DNA, several types of DNA can be used, such as plasmids, PCR products, or genomic DNA.

For more information, see Appendix E 'Generating Standard Curves' in the QuantiTect Probe PCR Handbook.

FAQ ID -1085

Rotor-Gene Kits are specifically developed for the Rotor-Gene Q PCR Cycler. The unique rotary system of the cycler combined with the kits’ proprietary buffer system enable ultrafast cycling. Rotor-Gene Kits do not contain ROX dye since no normalization to a passive reference is required. Also, Rotor-Gene Kits do not contain dUTP; therefore, UNG pretreatment is not possible.

FAQ ID -2119

Yes, probes and primers can be added to the QuantiTect Probe PCR- and the QuantiTect Probe RT-PCR Master Mix. This master mix can be stored at -20°C for up to 50 days, with a maximum of six freeze-thaw cycles (tested in our labs).

The master mix must be stored protected from light to avoid bleaching of the fluorescent probe. However, the QuantiTect RT Mix should be stored separately at –20°C, because the reverse transcriptases are sensitive to freezing in an aqueous environment. The QuantiTect RT Mix should be added to this master mix just prior to adding the template RNA and placing the reactions into the thermal cycler.

FAQ ID -584

We have compared QuantiFast Kits and QuantiTect Kits using around 30 different assays (using both SYBR Green and Probe detection for each assay).

QuantiFast Kits gave identical or sometimes better Ct values than QuantiTect Kits (except for very long amplicons). Therefore, scientists switching from QuantiTect to QuantiFast Kits can, in most cases, obtain comparable results.

FAQ ID -1441

Yes - dTTP in the QuantiTect Master Mix in QuantiTect SYBR Green and Probe PCR Kits is partially substituted by dUTP, therefore allowing treatment with uracil-N-glycosylase.

However, only heat-labile UNG can be used for one-step RT-PCR with the QuantiTect Probe RT-PCR and SYBR Green RT-PCR Kits. This is because heat stable UNG (isolated from E. coli) will destroy any cDNA synthesized during the RT step at 50°C. Heat-labile UNG will have lost any activity after a few minutes of the RT-step and therefore cannot interfere with cDNA synthesis.

For heat-stable uracil-N-glycosylase, please use the QuantiTect SYBR Green PCR +UNG (cat. no. 204163) or QuantiTect Probe PCR +UNG Kits (cat. no. 204363). Both kits include heat-stabile uracil-N-glycosylase. Although QIAGEN UNG is inactivated by incubation at 95°C for 15 minutes, the enzyme may exhibit residual activity at lower temperatures due to refolding. Therefore, it is recommended to perform subsequent PCR using a temperature of 55°C or above for the annealing step.

FAQ ID -564

LightCycler: We do not recommend performing real-time, multiplex PCR using TaqMan® probes or QuantiTect Assays on the LightCycler system due to the limitations of its optical detection system.
LightCycler 2.0: Information on using the QuantiTect Multiplex PCR NoROX Kit with the LightCycler 2.0 system is provided in the QuantiTect Multiplex PCR NoROX Handbook. You will find the recommendation for the LightCycler 2.0 system in the chapter 'Important Notes' under "Suitable combinations of reporter dyes".

See trademarks.

FAQ ID -717

Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.

Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (T_m) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer T_m, but approximately 3ºC lower than the specific amplicon T_m.

FAQ ID -9096

The emission maximum of Yakima Yellow (552 nm) is almost identical to that of the fluorescent dye VIC. Therefore, the channel and filter settings for VIC can also be used for Yakima Yellow.

FAQ ID -540

The melting temperature of a PCR product depends on the salt concentration of the buffers used for amplification. Buffer compositions differ between QuantiTect SYBR Green PCR Kits and Roche kits, resulting in different PCR product melting temperatures.

FAQ ID -1084