QuantiFast Multiplex RT-PCR Kits

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

Features

  • 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

Products

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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QuantiFast Multiplex RT-PCR +R Kit (400)

Cat. No. / ID: 204954

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
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QuantiFast Multiplex RT-PCR +R Kit (2000)

Cat. No. / ID: 204956

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
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QuantiFast Multiplex RT-PCR Kit (400)

Cat. No. / ID: 204854

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

Product Details

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.

 

IMPORTANT NOTE: The QuantiFast Multiplex RT-PCR Kit (400) and the QuantiFast Multiplex RT-PCR +R Kit (400) are phasing-out until June 30, 2021, and afterward, available only until stocks last. Visit the product page of the successor kit to view improved features or to request a trial kit.

 

The QuantiFast Multiplex RT-PCR +R Kit (2000) will remain available.

 

For more information and FAQs on this transition, visit: www.qiagen.com/PCRresource.

Performance

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").

See figures

Principle

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.
See figures

Procedure

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

Applications

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.

Supporting data and figures

Specifications

FeaturesSpecifications
applicationsReal-time quantification of RNA targets in a multiplex format
reactiontypeReal-time one-step RT-PCR
singleormultiplexMultiplex
sybrgreeniorsequencespecificprobesSequence-specific probes
realtimeorendpointReal-time
thermalcyclerReal-time cyclers dedicated for multiplex PCR (e.g., most Applied Biosystems real-time PCR cyclers, Roche LightCycler 480, and Bio-Rad iCycler iQ)
sampletargettypeRNA
withorwithoutroxAvailable with ROX in master mix or with ROX as separate vial

Resources

Quick-Start Protocols (2)
Kit Handbooks (1)
For quantitative, multiplex, real-time one-step RT-PCR with fast cycling using sequence-specific probes
Gene Expression Analysis (1)

FAQ

What is the maximum number of targets that can be amplified per reaction with the QuantiFast Multiplex RT-PCR Kit?

We have successfully tested up to 4 targets per reaction with the QuantiFast Multiplex RT-PCR Kit. A higher degree of multiplexing is achievable only on a limited number of PCR instruments (e.g., LightCycler 2.0). For these rare applications, further optimization of primer–probe concentration, cycling protocol, etc., might be needed.

 

 

FAQ ID -2145
Do you have any information or guidelines regarding the choice of reference genes for real-time PCR?

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 [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
Why do replicates in real-time PCR have different plateau heights?

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
Why are the denaturation and annealing/extension times for QuantiFast Multiplex RT-PCR Kits much shorter than those for QuantiFast Multiplex PCR Kits?

The QuantiFast Buffer in the QuantiFast Multiplex RT-PCR Kit has been further optimized to enable fast multiplex one-step RT-PCR.

 

 

 

FAQ ID -2147
How important is the RNA purification process, for obtaining reliable qRT-PCR results?

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
What is the threshold cycle or Ct value?
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
What is the detection limit of the Rotor-Gene and QuantiFast Multiplex RT-PCR Kits?

The Rotor-Gene and QuantiFast Multiplex RT-PCR Kits allow reliable detection down to 10 target copies. Detection of lower copy numbers down to single copy level may also be possible; however, this depends on the stochastics when working with highly diluted samples. Additional optimization of primer/probe design is usually required.

 

 

FAQ ID -2144
Why does my realtime PCR assay quality decrease over time?
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
Why should DNA or cDNA targets be less than 250 bp long for real-time PCR?

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
What are the main differences between Rotor-Gene and QuantiTect or QuantiFast PCR Kits?

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