QuantiFast Multiplex PCR Kits
For fast, multiplex, real-time PCR and two-step qRT-PCR using sequence-specific probes
For fast, multiplex, real-time PCR and two-step qRT-PCR using sequence-specific probes
Cat. No. / ID: 204654
Cat. No. / ID: 204756
QuantiFast Multiplex PCR Kits enable fast and reliable quantification of up to 4 cDNA or gDNA targets in a single tube by multiplex, real-time PCR or two-step RT-PCR. Q-bond technology and an optimized master mix promote fast multiplex real-time 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 PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiFast Multiplex PCR +R Kit for all other cyclers. For convenience, the master mix can be stored at 2–8°C.
IMPORTANT NOTE: As announced earlier, the production of the QuantiFast kits has been discontinued since mid-2021. Hence, these products will be available only until stocks last. Visit the product page of the successor kit to view improved features or to request a trial kit.
For more information and FAQs on this transition, visit: www.qiagen.com/PCRresource.
The special master mix supplied with QuantiFast Multiplex PCR Kits allows rapid setup of multiplex reactions and delivers successful results at the first attempt, providing multiplex PCR data that are comparable with singleplex PCR data (see figure " Comparable results in triplex and singleplex PCR"). The kits can clearly distinguish between small differences in the amount of template. Even with twofold differences in template amount, QuantiFast Multiplex PCR Kits provide accurate quantification of targets of widely differing abundance (see figure " Linear CT values over twofold decreases in template").
QuantiFast Multiplex PCR Kits reduce PCR run times by up to 50%, allowing you to get results significantly faster (see figure " Significantly reduced PCR times"). As little as 10 copies of target can be detected in just 60 minutes (see figure " Sensitive duplex PCR and a wide dynamic range"). You can also greatly increase your sample throughput or efficiently share a cycler with other users. Fast results in multiplex, real-time PCR of up to 4 targets are achieved without compromising performance (see figure " Uncompromised sensitivity").
QuantiFast Multiplex 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 PCR Buffer includes a balanced combination of K+ and NH4+ ions as well as the unique synthetic Factor MP, which together promote stable and efficient annealing of primers and probes to the nucleic acid template, enabling high PCR efficiency (see figure " Unique PCR buffer"). In addition, HotStarTaq Plus DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.
|HotStarTaq Plus DNA Polymerase||5 min activation at 95ºC||Set up of qPCR reactions at room temperature|
|QuantiFast Multiplex 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 Multiplex PCR Kits contain ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. Simply add template DNA 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.
|ROX dye||Kit||Compatible cyclers|
|Supplied in master mix||QuantiFast Multiplex PCR Kit||All cyclers from Applied Biosystems except Applied Biosystems 7500|
|Supplied in separate tube||QuantiFast Multiplex PCR +R Kit||Applied Biosystems 7500 and cyclers from
Bio-Rad, Cepheid, Eppendorf, QIAGEN, Roche, Agilent, and other suppliers
For optimal results in real-time two-step RT-PCR, we recommend synthesizing cDNA using the QuantiTect Reverse Transcription Kit, which provides fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination.
QuantiFast Probe Assays are predesigned, genomewide assays that use hydrolysis, probe-based detection. They are delivered with the QuantiFast Multiplex PCR Kit for guaranteed results in duplex, two-step real-time RT-PCR.
QuantiFast Multiplex PCR Kits can be used for multiplex gene expression analysis of cDNA or gDNA targets on any real-time cycler. This includes instruments from Agilent, Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, and Roche. For the Rotor-Gene Q and other Rotor-Gene cyclers, we recommend using the Rotor-Gene Multiplex PCR Kit, which has been specially developed for fast cycling on these instruments.
|Applications||Quantification of cDNA or genomic DNA targets in a multiplex real-time PCR|
|Thermal cycler||Real-time cyclers dedicated for multiplex PCR (e.g., most Applied Biosystems real-time PCR cyclers, Roche LightCycler® 480, iCycler iQ®, Rotor-Gene)|
|Real-time or endpoint||Real-time|
|Sample/target type||cDNA, DNA|
|SYBR Green I or sequence-specific probes||Sequence-specific probes|
|Reaction type||Real-time two-step RT-PCR|
|Single or multiplex||Multiplex|
|With or without ROX||Available with ROX in master mix and with ROX as separate vial|
No, that is not necessary. Simply use the primer concentrations specified in the protocols in the handbook supplied with your QuantiFast Multiplex PCR Kit.
QuantiFast Probe Assays are only sold in combination with optimized real-time RT-PCR master mixes and cannot be sold as stand-alone assays.
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.
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 : 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 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.
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.
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".
Our unique multiplex PCR buffer system with ammonium and potassium ions and Factor MP has been further optimized in QuantiFast and Rotor-Gene Kits. We have also discovered Q-Bond, a buffer component which supports the rapid formation of the polymerase–primer–template complex, leading to reduced annealing times.
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.
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.
No, the QuantiFast Multiplex PCR Kit cannot be used for 4plex real-time PCR on the ABI PRISM 7000, 7700, or 7900 instruments. Due to hardware limitations, the maximum capacity of these real-time cyclers is triplex PCR.