QuantiFast Multiplex RT-PCR Kits

For fast, multiplex, one-step qRT-PCR using sequence-specific probes for gene expression analysis

  • Sensitive detection of multiple RNA targets in 1 well
  • Faster results with time savings of up to 50%
  • Successful multiplex RT-PCR without the need for optimization
  • Precise discrimination of small differences in target amount
  • Detection of reference gene and up to 3 targets in the same tube

QuantiFast Multiplex RT-PCR Kits enable fast and reliable quantification of up to 4 RNA targets in a single tube by multiplex, real-time one-step RT-PCR. A special blend of reverse transcriptases delivers fast and efficient cDNA synthesis. Q-bond technology and an optimized master mix promote fast, multiplex real-time RT-PCR, not only on fast cyclers with short ramping times, but also on standard cyclers. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization. Two kit formats are available: the QuantiFast Multiplex RT-PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiTect Multiplex RT-PCR +R Kit for all other cyclers. For convenience, the master mix can be stored at 2–8°C.

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QuantiFast Multiplex RT-PCR Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Multiplex RT-PCR Master Mix (with ROX dye), 100 µl QuantiFast RT Mix, 2 x 2 ml RNase-Free Water
QuantiFast Multiplex RT-PCR +R Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Multiplex RT-PCR Master Mix (without ROX dye), 100 µl QuantiFast RT Mix, 210 µl ROX Dye Solution, 2 x 2 ml RNase-Free Water
QuantiFast Multiplex RT-PCR +R Kit (2000)
For 2000 x 25 µl reactions: 25 ml 2x QuantiFast Multiplex RT-PCR Master Mix (without ROX dye), 500 µl QuantiFast RT Mix, 1.05 ml ROX Dye Solution, 1 x 20 ml RNase-Free Water

QuantiFast Multiplex RT-PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

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Significantly reduced RT-PCR times.|Reliable relative quantification.|Comparable results in triplex and singleplex RT-PCR.|Uncompromised sensitivity in 4-plex RT-PCR.|Fast primer annealing.|QIAGEN multiplex kits.|Unique buffer promotes stable and efficient primer annealing.|
QuantiFast Multiplex Kits reduce total RT-PCR run time by up to 50% in real-time one-step RT-PCR (40 cycles run; comparison with QuantiTect Multiplex Kits). I: iCycler iQ; L1: LightCycler 480; L2: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500 Fast System; M: Mx3005P.
|Cell line X was treated with one of two compounds (Y or Z) or left untreated (U). RNA was purified and duplex, real-time one-step RT-PCR was carried out on the Applied Biosystems 7500 Fast System using the QuantiFast Multiplex RT-PCR +R Kit and TaqMan Gene Expression Assays for myogenin and GAPDH. For each treatment, 5 independent experiments were performed. [A] Changes in myogenin expression were detected with high accuracy and reproducibility (green curves). The expression of the housekeeping gene GAPDH was similar in all experiments (blue curves), allowing normalization of myogenin expression levels using the ΔΔCT method of relative quantification. [B] The fold changes in normalized myogenin expression level relative to that in untreated cells were consistent between experiments, as indicated by the small error bars. (Data kindly provided by Angelika Meyer, Novartis Pharma AG, Basel, Switzerland).|Triplex and singleplex, real-time one-step RT-PCR were carried out on the LightCycler 480 using the QuantiFast Multiplex RT-PCR +R Kit and self-designed TaqMan assays for [A] RPS27A (a ribosomal protein), [B] GAPDH (a housekeeping gene), and [C] UBC (a housekeeping gene). The template was Ramos cell line RNA (10 ng, 1 ng, or 0.1 ng), and reactions were run in duplicate. The comparable CT values for triplex PCR (colored curves) and singleplex PCR (gray curves) and [D] the high PCR efficiencies demonstrate the reliability of triplex PCR with the QuantiFast Multiplex RT-PCR +R Kit when analyzing targets of differing abundance.|4-plex, real-time one-step RT-PCR was carried out on the Mx3005P using the QuantiFast Multiplex RT-PCR +R Kit and Primer Express designed TaqMan assays. Duplicate reactions were run using 10 ng RNA from Ramos cells as template. All 4 targets, which varied greatly in abundance, were reproducibly detected.|[A] Q-Bond in QuantiFast Buffer allows the DNA polymerase and primer to bind as a single complex, reducing the annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing annealing time.|QuantiFast Multiplex RT-PCR Kits provide a simple procedure for quantitative, multiplex, real-time PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, Mg2+, and Taq DNA polymerase, even for difficult assays (e.g., assays in which the copy number of the target gene is much smaller than that for the reference gene).|[A] NH4+ ions prevent nonspecific primers from annealing to the template. [B] Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. Together with K+ and other cations, Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq Plus DNA Polymerase.|

QuantiFast Multiplex RT-PCR Kits reduce RT-PCR run times by up to 50%, allowing you to get results significantly faster (see figure "Significantly reduced RT-PCR times"). You can also greatly increase your sample throughput or efficiently share a cycler with other users. Amplifying control and target genes in the same reaction, instead of in separate reactions, increases the reliability of gene quantification by minimizing handling errors (see figure "Reliable relative quantification"). The special master mix supplied with QuantiFast Multiplex RT-PCR Kits allows rapid setup of multiplex reactions and delivers successful results at the first attempt, providing multiplex RT-PCR data that are comparable with singleplex RT-PCR data (see figure "Comparable results in triplex and singleplex RT-PCR").

QuantiFast Multiplex RT-PCR Kits can clearly distinguish between small differences in the amount of template. Even with two-fold differences in template amount, the kits provide accurate quantification of targets of widely differing abundance. Fast results in multiplex, real-time RT-PCR of up to 4 targets are achieved without compromising performance (see figure "Uncompromised sensitivity in 4-plex RT-PCR").


QuantiFast Multiplex RT-PCR Kits deliver highly sensitive and rapid results over a wide dynamic range on both standard and fast cyclers without optimization (see flowchart "QIAGEN multiplex kits"). The specially developed fast PCR buffer contains the novel additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure "Fast primer annealing").

Amplifying reference and target genes in the same reaction instead of in separate reactions increases the reliability of gene quantification by minimizing handling errors. The QuantiFast Multiplex RT-PCR Buffer includes a balanced combination of K+ and NH4+ ions to promote specific primer annealing, while unique Factor MP stabilizes specifically bound primers (see figure "Unique PCR buffer"). In addition, an optimized mix of reverse transcriptases provides efficient cDNA synthesis in just 20 minutes, while HotStarTaq Plus DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

Components of 2x QuantiFast Multiplex RT-PCR Kit
Component Features Benefits
HotStarTaq Plus DNA Polymerase 5 min activation at 95ºC Set up of qPCR reactions at room temperature
QuantiFast Multiplex RT-PCR Buffer Balanced combination of NH4+ and K+ ions Specific primer annealing ensures reliable PCR results
Synthetic Factor MP Reliable multiplexing analysis of up to 4 genes in the same tube
Unique Q-Bond additive Faster PCR run times, enabling faster results and more reactions per day
ROX dye Normalizes fluorescent signals on Applied Biosystems and, optionally, Agilent instruments Precise quantification on cyclers that require ROX dye. Does not interfere with PCR on any real-time cycler
QuantiFast RT Mix Special blend of reverse transcriptases with high affinity for RNA RNA can be transcribed in just 20 minutes, even through complex secondary structures
*  Also contains a dNTP mix (dATP, dCTP, dGTP, and dTTP).
 ROX dye is either present in the master mix or as a separate solution.

QuantiFast Multiplex RT-PCR Kits contain ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. Simply add template RNA and primer-probe sets to the master mix and follow the protocol in the handbook to get fast and reliable results on any real-time cycler. Kits are available with or without ROX passive reference dye in the master mix, enabling use on virtually any real-time cycler (see table).  Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.

Choosing the right QuantiFast Multiplex RT-PCR Kit
ROX dyeKit Compatible cyclers
Supplied in master mix QuantiFast Multiplex RT-PCR Kit All cyclers from Applied Biosystems except Applied Biosystems 7500
Supplied in separate tube QuantiFast Multiplex RT-PCR +R Kit Applied Biosystems 7500 and cyclers from
Bio-Rad, Cepheid, Eppendorf, QIAGEN, Roche, Agilent, and other suppliers

QuantiFast Multiplex RT-PCR Kits can be used for multiplex gene expression analysis of RNA targets on any real-time cycler. This includes instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene Multiplex RT-PCR Kit, which has been specially developed for fast cycling on these instruments.

Applications Real-time quantification of RNA targets in a multiplex format
Reaction type Real-time one-step RT-PCR
Real-time or endpoint Real-time
Sample/target type RNA
Single or multiplex Multiplex
SYBR Green I or sequence-specific probes Sequence-specific probes
Thermal cycler Real-time cyclers dedicated for multiplex PCR (e.g., most Applied Biosystems real-time PCR cyclers, Roche LightCycler 480, and Bio-Rad iCycler iQ)
With or without ROX Available with ROX in master mix or with ROX as separate vial

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Kit Handbooks
For quantitative, multiplex, real-time one-step RT-PCR with fast cycling using sequence-specific probes
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