For fast, real-time PCR and two-step qRT-PCR using sequence-specific probes
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
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.
For 2000 x 25 µl reactions: 25 ml 2x QuantiFast Probe PCR Master Mix (without ROX dye), 1.05 ml ROX Dye Solution, 20 ml RNase-Free Water
QuantiFast Probe PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
Significantly reduced PCR times.
QuantiFast Probe Kits reduce total PCR run time by up to 60% in real-time two-step RT-PCR on standard cyclers (40 PCR cycles carried out; comparison with a standard QIAGEN real-time PCR kit). L: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500; A3: ABI PRISM 7000.
Wide dynamic range and high sensitivity.
The QuantiFast Probe PCR +ROX Vial Kit provided accurate gene expression analysis from low to high template amounts with a PCR efficiency of 93% in two-step PCR on the Mastercycler ep realplex. Duplicate reactions (25 μl volume) were performed using 10-fold dilutions of human leukocyte cDNA and a Primer Express designed TaqMan assay for ubiquitin (a regulatory protein). NTC: no template control.
Faster results without compromising sensitivity.
Reactions were run in duplicate using 10-fold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a TaqMan gene expression assay for IL1RN (a cytokine) on the ABI PRISM 7900. The QuantiFast Kit showed greater sensitivity than the standard cycling kit from Supplier AII (which was used according to the standard cycling protocol), providing much lower CT values and transcript quantification from down to 10 pg cDNA.
Sensitive two-step RT-PCR.
Reactions were run in triplicate using 10-fold dilutions of human leukocyte cDNA (100 ng to 0.01 ng) and a Primer Express designed TaqMan assay for IL8 (a chemokine) on the ABI PRISM 7900. [A] The QuantiFast Kit showed greater sensitivity than [B] the kit from Supplier I (which was used according to the standard cycling protocol), providing lower CT values and transcript quantification from down to 0.01 ng cDNA.
Detection over 7 Logs of template.
Duplicate two-step RT-PCR reactions were run using a set of primers and FRET probes for b-2 microglobulin from Supplier R on the LightCycler 2.0. The amplification plots for the QuantiFast Kit show the kit's wide dynamic range.
Specific primer annealing.
A balanced combination of KCl and (NH4)2SO4 promotes specific annealing of primers and probes to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing annealing of primers and probes. NH4+ destabilizes weak hydrogen bonds between mismatched bases.
Performance
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
Triplicate reactions were run using a Primer Express designed TaqMan® assay for IL8 (a chemokine). The QuantiFast Kit provided lower CT values than the kit from Supplier BV (which was used according to a fast-cycling protocol), allowing transcript quantification from just 1 ng cDNA.
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%
Duplicate reactions were run using a set of primers and FRET probes for β-2 microglobulin from supplier R. The QuantiFast Kit provided lower CT values and higher PCR efficiency that the kit from Supplier R (which was used according to the fast cycling protocol ).
Principle
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
* Also contains dNTP mix (dATP, dCTP, dGTP, and dTTP). † ROX dye is either present in the master mix or as a separate solution.
Procedure
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.
Applications
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.
Features
Specifications
Applications
Probe-based, real-time PCR, two-step RT-PCR
Reaction type
Real-time and two-step RT-PCR
Real-time or endpoint
Real-time
Sample/target type
cDNA, DNA
Single or multiplex
Single
SYBR Green I or sequence-specific probes
Sequence-specific probes
Thermal cycler
All real-time cyclers (e.g. Roche LightCycler®, Corbett Rotor-Gene, Applied Biosystems)
With or without ROX
Available with ROX in master mix and 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)
