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.
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
Significantly reduced RT-PCR times.
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.
Reliable relative quantification.
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).
Uncompromised sensitivity in 4-plex RT-PCR.
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.
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
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
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
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.
Real-time quantification of RNA targets in a multiplex format
Real-time one-step RT-PCR
Real-time or endpoint
Single or multiplex
SYBR Green I or sequence-specific probes
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
For 100 x 50 µl reactions: QIAGEN OneStep RT-PCR Enzyme Mix (1 x 200 µl), 5x QIAGEN OneStep RT-PCR Buffer (1 x 1 ml), dNTP Mix (1 x 200 µl, 10 mM each), 5x Q-Solution (1 x 2 ml), RNase-Free Water (2 x 1.9 ml)