QuantiFast Probe PCR Kits

For fast, real-time PCR and two-step qRT-PCR using sequence-specific probes


QuantiFast Probe PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
QuantiFast Probe PCR Kit (2000)

Cat. No. / ID: 204256

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.   For 2000 x 25 µl reactions: 25 ml 2x QuantiFast Probe PCR Master Mix (contains ROX dye), 20 ml RNase-Free Water
This kit is being phased out. We recommend switching to the QuantiNova successor product. For more information and FAQs on this transition, visit: www.qiagen.com/PCRresource.


  • Sensitive detection of even low copy targets
  • Accurate detection of a wide range of template amounts
  • Faster results with a time savings of up to 60%
  • Optimized, ready-to-use master mix for reliable results without optimization
  • One cycling protocol for all standard and fast cyclers

Product Details

QuantiFast Probe PCR Kits deliver fast and sensitive quantification of gDNA and cDNA targets by real-time PCR and two-step RT-PCR using sequence-specific probes. Q-bond technology and an optimized master mix enable shorter real-time PCR run times, 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 Probe PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiFast Probe PCR +ROX Vial 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.


QuantiFast Probe PCR Kits deliver highly sensitive results, outperforming other real-time PCR kits (see table "Sensitive two-step RT-PCR on the iCycler iQ" and figure and  Sensitive two-step RT-PCR). PCR run times are reduced by up to 60% (see figure " Significantly reduced PCR times"), allowing you to achieve fast PCR results without compromising on PCR performance (see figure " Faster results without compromising sensitivity"). You can also greatly increase your sample throughput or efficiently share a cycler with other users.

Sensitive two-step RT-PCR on the iCycler iQ
  Mean CT value  
Human leukocyte cDNA (ng) QuantiFast Probe PCR +ROX Vial Kit Kit from Supplier BV
100 29.13 31.13
10 32.50 34.43
1 36.15 40.00
No template control 40.00 40.00


QuantiFast Probe PCR Kits allow accurate quantification over a wide dynamic range (see figure " Wide dynamic range and high sensitivity"). Template dilutions of up to 7 logs can be reliably detected (see table "Detection over 7 logs of template in two-step RT-PCR on the LightCycler 2.0 " and figure " Detection over 7 logs of template").

Detection over 7 logs of template in two-step RT-PCR on the LightCycler 2.0
  Mean CT value  
Human leukocyte cDNA QuantiFast Probe PCR +ROX Vial Kit Kit from Supplier R
100 ng 11.99 14.56
10 ng 15.54 17.99
1 ng 19.22 22.16
100 pg 22.63 25.14
10 pg 26.43 30.15
1 pg 30.02 34.43
100 fg 33.42 37.10
No template control 40 40.00
PCR efficiency 90% 80.1%
See figures


QuantiFast Probe PCR Kits deliver highly sensitive and rapid results over a wide dynamic range on both standard and fast cyclers. The kits are designed for use with all types of sequence-specific probes, including hydrolysis probe detection (e.g., TaqMan and other dual-labeled probes) and FRET probes. A specially developed, fast PCR buffer contains the novel additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure " Fast primer annealing"). The buffer also contains a balanced combination of K+ and NH4+ ions, which promote specific primer annealing, enabling high PCR specificity and sensitivity (see figure " Specific primer annealing"). In addition, HotStarTaq Plus DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

Components of 2x QuantiFast Probe PCR Kit*
Component Features and benefits Benefits
HotStarTaq Plus DNA Polymerase 3 min activation at 95ºC Set up of qPCR reactions at room temperature
QuantiFast Probe PCR Buffer Balanced combination of NH4+ and K+ ions Specific primer annealing ensures reliable PCR results
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
See figures


QuantiFast Probe PCR Kits contain ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. Simply add template DNA, primers, and probe 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 Probe PCR Kit
ROX dye Kit Compatible cyclers
Supplied in master mix QuantiFast Probe PCR Kit All cyclers from Applied Biosystems except Applied Biosystems 7500
Supplied in a separate tube QuantiFast Probe PCR +ROX Vial Kit Applied Biosystems 7500, and cyclers from Agilent, Bio-Rad, Cepheid, QIAGEN, Eppendorf, Roche, and other suppliers

For optimal results in real-time two-step RT-PCR, we recommend synthesizing cDNA using the QuantiTect Reverse Transcription Kit. The kit 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 Probe PCR Kit for guaranteed results in singleplex, two-step qRT-PCR.


QuantiFast Probe PCR Kits can be used for probe-based gene expression analysis of cDNA targets and quantitative gDNA analysis 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 Probe PCR Kit, which has been specially developed for fast cycling on these instruments.

Supporting data and figures


ApplicationsProbe-based, real-time PCR, two-step RT-PCR
Sample/target typecDNA, DNA
Real-time or endpointReal-time
Reaction typeReal-time and two-step RT-PCR
Single or multiplexSingle
With or without ROXAvailable with ROX in master mix and with ROX as separate vial
SYBR Green I or sequence-specific probesSequence-specific probes
Thermal cyclerAll real-time cyclers (e.g. Roche LightCycler®, Corbett Rotor-Gene, Applied Biosystems)


Safety Data Sheets (1)
Quick-Start Protocols (2)
Kit Handbooks (1)
For fast, quantitative, real-time PCR and two-step RT-PCR using sequence-specific probes


Can fast cycling be carried out with QuantiFast Probe PCR Kits and FRET probes?

Yes, QuantiFast Probe PCR Kits allow the use of FRET probes, but only in 3-step cycling with a shorter initial activation step and a shorter reverse-transcription step.

A protocol is available in the QuantiFast Probe PCR Handbook.



FAQ ID -1433
Can QuantiFast PCR Kits be used on real-time PCR instruments without fast cycling options?

Yes, QuantiFast Kits can also be run on a qPCR cycler without fast cycling options. You cannot achieve rapid ramping rates, but you can still take advantage of the combined annealing/extension step and the reduced denaturation and annealing/extension times offered by QuantiFast Kits.

You will be able to obtain your PCR results in a much shorter time.


FAQ ID -1428
Is the master mix of QuantiFast Kits for real-time PCR aliquoted into several tubes to prevent cross-contamination?

QuantiFast Kits for 400 x 25 µl reactions contain a master mix that is aliquoted into 3 separate tubes.

QuantiFast Kits for 2000 x 25 µl reactions provide one tube containing 25 ml master mix to offer a cost-effective solution for higher throughput experiments.



FAQ ID -1697
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 is the reaction volume for QuantiFast PCR Kits lower than that for QuantiTect PCR Kits?

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
Do you offer trial-kit sizes for the new QuantiFast Kits?

Yes, the QuantiFast SYBR Green PCR Kit and QuantiFast Probe PCR Kits are available for 80 x 25 µl reactions. This trial-kit size is not available for QuantiFast RT-PCR Kits.


FAQ ID -1429
How do Rotor-Gene Probe Kits compare with QuantiFast Probe Kits?

Rotor-Gene Probe Kits are specially developed for Rotor-Gene cyclers. The unique rotary system of the cyclers combined with the kits’ proprietary buffer system enable ultrafast cycling. Rotor-Gene Probe Kits do not contain ROX dye.


FAQ ID -2125
Is the QuantiFast Probe PCR +ROX Vial Kit compatible with the ViiA7 cycler from Applied Biosystems?
We got comparable results on the ABI 7500 and ViiA7 using the QuantiFast Probe PCR +ROX Vial Kit master mix with low ROX concentration (ROX added to NoROX master mix) and the protocol for the ABI 7500.
FAQ ID -2651
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
How should fluorescent labeled probes be stored?

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
Do you have information on the use of recombinant DNA and RNA as absolute standards for realtime RT-PCR?

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
Can I do singleplex PCR with the QuantiFast Probe Assays?
Yes, for 2-step RT-PCR, the QuantiFast Probe PCR Kit can be used, for 1-step RT-PCR, the QuantiFast Probe RT-PCR Plus Kit is combined with the assays.
FAQ ID -2362
How many reactions can I perform with the new QuantiFast Kits for real-time PCR?

Compared with QuantiTect Kits, the recommended reaction volume for QuantiFast Kits is reduced from 50 µl to 25 µl (96-well block cyclers), and from 20 µl to 10 µl (384-well block cyclers).

The volume of master mix remains the same, which means that QuantiFast Kits offer twice the number of reactions as QuantiTect Kits. However, for LightCycler instruments, the recommended reaction volume remains the same (20 µl).

FAQ ID -1425
Are TaqMan® Gene Expression Assays from Applied Biosystems compatible with QuantiFast Probe PCR Kits?

Yes, use the ABI gene expression assays at a final concentration of 1x with the QuantiFast Probe PCR Kits.

See trademarks

FAQ ID -1452
Why is the storage time for QuantiFast PCR Kits shorter than that for QuantiTect PCR Kits?

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
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 QuantiFast Kits for real-time PCR?

The QuantiFast SYBR Green PCR and QuantiFast Probe 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, it depends on the stochastics when working with highly diluted samples. Additional optimization of primer/probe design is usually required.

FAQ ID -1432
How do you achieve fast cycling, yet still deliver the same performance in PCR as that achieved with standard cycling?

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.

FAQ ID -1430
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
Which real-time PCR kits are recommended downstream of the QuantiTect Whole Transcriptome Kit?

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


FAQ ID -1592
Do special settings have to be used for QuantiFast PCR Kits on the Eppendorf Mastercycler ep realplex?

No. Optimized thermal cycling programs for use with QuantiFast Kits and a Program Selection Guide are available.

To install these programs on your Mastercycler ep realplex, contact your Eppendorf sales representative or visit our QIAGEN/Eppendorf Alliance page.


FAQ ID -1437
Can the QuantiFast Probe PCR +ROX Vial Kit be used on the ABI PRISM 7900 and Applied Biosystems 7500 cyclers?

The ABI PRISM 7900 requires a high ROX concentration, which cannot be achieved with the 50x ROX dye solution supplied in the QuantiFast Probe PCR +ROX Vial Kit. However, this kit provides the optimal ROX concentration for the Applied Biosystems 7500.


FAQ ID -1555
How much time will be saved when switching from standard cycling to fast cycling with QuantiFast Kits?

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
Are there problems with the default threshold setting on the Applied Biosystems 7500 when using QuantiFast Probe PCR Kits?

No. We recommend using QuantiFast Probe +ROX Vial Kits: the master mix does not contain ROX dye, but the kit is supplied with a separate tube of ROX dye. The kits allow to adjust the ROX concentration in the master mix according to your cycler’s requirements.

Please follow specific recommendations in the QuantiFast Probe PCR Handbook.


FAQ ID -1702
What is Q-Bond used in the QIAGEN Fast Cycling PCR and QuantiFast Kits?

The Q-Bond Molecule, present in the PCR Buffer of the QIAGEN Fast Cycling PCR Kit and the QuantiFast Kits, dramatically increases the binding affinity of DNA polymerase to single-stranded DNA, thereby facilitating the reduction of annealing time to just a few seconds. It is a non-protein PCR component. Unfortunately, all further information on this molecule is proprietary.

FAQ ID -1554
Why is the activation time for HotStarTaq Plus Polymerase in the QuantiFast SYBR Green Kits different from that for QuantiFast Probe Kits?

The activation time for HotStarTaq Plus DNA Polymerase used in the QuantiFast SYBR Green PCR Kits is longer than that for QuantiFast Probe PCR Kits. This is due to differences in buffer composition. Buffer components such as salts and additives influence the time required for enzyme activation.


FAQ ID -1449
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 should I use as a standard for absolute quantification in real-time PCR?

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
Can 2 µl reaction volumes be used with QuantiFast PCR Kits?

We recommend a reaction volume of 10 µl when using 384-well blocks with QuantiFast PCR Kits. If reducing the reaction volume to 2 µl, results will vary depending on the real-time cycler used.

Please contact QIAGEN Technical Services for more information.

FAQ ID -1440
Why is the QuantiFast denaturation step different for PCR and RT-PCR runs in the two-step protocol for the ABI 7500 and other cyclers?

This is due to differences in composition between PCR and RT-PCR buffers. QuantiFast PCR Buffers are optimized for fast amplification with shortest possible PCR steps, while QuantiFast RT-PCR Buffers are optimized for reverse transcription and subsequent amplification.

FAQ ID -1442
What QuantiFast Kit should be used on the Eppendorf Mastercycler ep realplex?

The Mastercycler ep realplex does not require ROX dye. You can use QuantiFast Probe PCR +ROX Vial Kits (no ROX dye in the master mix), QuantiFast SYBR Green PCR and QuantiFast SYBR Green RT-PCR Kits (ROX dye in the master mix does not interfere with real-time quantification).


FAQ ID -1436
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
Does the master mix in the QuantiFast Kits contain dUTP to allow UNG treatments?

No. The master mix in QuantiFast PCR Kits contains only dTTP. To perform a UNG treatment, we recommend using QuantiTect Kits.


FAQ ID -1431
How do QuantiFast PCR Kits compare to QuantiTect PCR Kits for quantitative real-time PCR?

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
Is it necessary to perform calibration steps for the use of the MAX dye in duplex RT-PCR experiments on different cyclers?

It is not necessary to perform calibration steps with MAX dye.  For instruments from Applied Biosystems, simply use the VIC channel/filter for the detection of MAX. The emission maxima of MAX and VIC are very similar (557nm and 554nm), respectively. On the Rotor-Gene Q, use the yellow channel. On the BioRad CFX, use channel 2 for MAX detection. On the LightCycler 480, use the combination 523 nm (Excitation) / 568 nm (Emission).  MAX was chosen as a second dye label because VIC label is protected by ABI and HEX showed more crosstalk in our experiments.

FAQ ID -2373
Can I adjust the ROX concentration in the QuantiFast master mix?

The master mix in QuantiFast SYBR Green Kits contains an optimized concentration of ROX dye that works well with all cyclers.

QuantiFast Probe PCR Kits are available in two formats:

  • the QuantiFast Probe PCR Kit with master mix containing ROX dye
  • the QuantiFast Probe PCR +ROX Vial Kit with master mix not containing ROX dye, and a separate vial of ROX dye

We recommend using the latter with the Applied Biosystems 7500 Fast System. Use the ROX concentration indicated in the QuantiFast Probe PCR Kits handbook.

FAQ ID -1427
Can the QuantiFast Probe PCR +ROX Vial mastermix be stored long-term after adding ROX dye?

Yes, when working with the QuantiFast Probe PCR +ROX Vial Kit, please add the volume of ROX dye solution specified in the QuantiFast Probe PCR Handbook.

Master mix containing ROX dye can be stored for up to 2 months at 4°C.


FAQ ID -1435