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

  • Faster results with time savings of up to 60%
  • Sensitive detection of even low copy numbers
  • Accurate detection of a wide range of template amounts
  • Optimized, ready-to-use master mix for fast cycling
  • One cycling protocol for all standard and fast cyclers
QuantiFast Probe RT-PCR Kits deliver fast and sensitive real-time one-step RT-PCR quantification of RNA targets using probe-based detection. Q-bond technology and an optimized master mix enable shorter real-time RT-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. The kits are also supplied with an optimized RT mix for efficient cDNA synthesis in only 10 minutes. Two kit formats are available: the QuantiFast Probe RT-PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiFast Probe RT-PCR +ROX Vial Kit for all other cyclers. For convenience, the master mix can be stored at 2–8°C.
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QuantiFast Probe RT-PCR Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Probe RT-PCR Master Mix (contains ROX dye), 100 µl QuantiFast RT Mix, 2 x 2 ml RNase-Free Water
204454
875,00 €
QuantiFast Probe RT-PCR Kit (2000)
For 2000 x 25 µl reactions: 25 ml 2x QuantiFast Probe RT-PCR Master Mix (contains ROX dye), 0.5 ml QuantiFast RT Mix, 20 ml RNase-Free Water
204456
3.472,00 €
QuantiFast Probe RT-PCR +ROX Vial Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Probe RT-PCR Master Mix (without ROX dye), 210 µl ROX Dye Solution, 100 µl QuantiFast RT Mix, 2 ml RNase-Free Water
204554
875,00 €
QuantiFast Probe RT-PCR +ROX Vial Kit (2000)
For 2000 x 25 µl reactions: 25 ml 2x QuantiFast Probe RT-PCR Master Mix (without ROX dye), 1.05 ml ROX Dye Solution, 0.5 ml QuantiFast RT Mix, 20 ml RNase-Free Water
204556
3.472,00 €

QuantiFast Probe 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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Significantly reduced RT-PCR times.|Sensitive one-step RT-PCR.|Comparable dynamic range.|Faster results without compromising sensitivity.|Wide dynamic range and high sensitivity.|Specific primer annealing.|Fast primer annealing.|
QuantiFast Probe Kits reduce total RT-PCR run time by up to 60% in real-time one-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.
|Reactions were run in duplicate using 10-fold dilutions of human leukocyte RNA (100 ng to 0.1 ng) and a Primer Express designed TaqMan assay for IL12RBI (a type I transmembrane protein) on the Applied Biosystems 7500 Fast System. [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 0.1 ng RNA.|Ubiquitin expression in human leukocytes was analyzed using a Primer Express designed TaqMan assay. Two-step and one-step qPCR reactions were run in duplicate on the ABI PRISM 7900. [A] 10-fold cDNA dilutions (100 ng to 10 pg) were analyzed using the QuantiFast Probe PCR Kit. [B] 10-fold RNA dilutions (100 ng to 10 pg) were analyzed using the QuantiFast Probe RT-PCR Kit.|Reactions were run in triplicate on the ABI PRISM 7900 using 10-fold dilutions of human leukocyte RNA (100 pg to 0.01 pg) and a TaqMan gene expression assay for 28S rRNA. [A] The QuantiFast Kit showed greater sensitivity than [B] the standard-cycling kit from Supplier AII (which was used according to the standard cycling-protocol), providing much lower CT values and quantification from as little as 0.01 pg RNA.|Triplicate reactions were performed using 10-fold dilutions of human leukocyte RNA and a TaqMan assay for β-actin. The QuantiFast Probe RT-PCR Kit provided accurate gene expression analysis from 100 ng down to 1 pg of RNA template.|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.|[A] Q-Bond in QuantiFast Buffer increases the affinity of DNA polymerase for short single-stranded DNA, reducing primer annealing time to a few seconds. In addition, the unique buffer composition supports the melting of DNA, reducing denaturation and extension times. [B] Without Q-Bond, the primer and polymerase bind sequentially to the template, increasing primer annealing time.|
Performance

QuantiFast Probe RT-PCR Kits deliver highly sensitive results, outperforming other real-time RT-PCR kits (see figure "Sensitive One-step RT-PCR"). RT-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 RT-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. With QuantiFast Probe RT-PCR Kits, you follow the same procedure whether you work with a standard or fast cycler.

QuantiFast Probe RT-PCR Kits allow accurate quantification over a wide dynamic range (see figure "Comparable dynamic range"). Template dilutions of up to 6 logs can be reliably detected (see figure "Wide dynamic range and high sensitivity").

Principle

QuantiFast Probe RT-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. The optimized QuantiFast RT Mix enables cDNA synthesis in just 10 minutes and a specially developed fast RT-PCR buffer contains the novel additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure "Fast primer annealing"). The RT-PCR buffer also contains a balanced combination of K+ and NH4+ ions, which promote specific primer annealing and enable high RT-PCR specificity and sensitivity (see figure "Specific primer annealing"). In addition, HotStarTaq Plus DNA Polymerase requires only 5 minutes at 95°C for activation and provides a stringent hot start, preventing the formation of nonspecific products.  

Components of 2x QuantiFast Probe RT-PCR Kit*
Component Features and benefits Benefits
HotStarTaq Plus DNA Polymerase 5 min activation at 95ºC Set up of qPCR reactions at room temperature
QuantiFast Probe RT-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
QuantiFast RT Mix Separate solution added during PCR setup Fast cDNA synthesis in just 10 minutes
* 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 RT-PCR Kits contain ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. Simply add template RNA, 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 RT-PCR Kit
ROX dyeKit Compatible cyclers
Supplied in master mix QuantiFast Probe RT-PCR Kit All cyclers from Applied Biosystems except Applied Biosystems 7500
Supplied in separate tube QuantiFast Probe RT-PCR +ROX Vial Kit Applied Biosystems 7500 and cyclers from
Bio-Rad, Cepheid, Eppendorf, QIAGEN, Roche, Agilent, and other suppliers
Applications

QuantiFast Probe RT-PCR Kits can be used for probe-based 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 Probe RT-PCR Kit, which has been specially developed for fast cycling on these instruments.

Feature
Specifications
Applications Probe-based, real-time RT-PCR
Reaction type Real-time one-step RT-PCR
Real-time or endpoint Real-time
Sample/target type RNA
Single or multiplex Single
SYBR Green I or sequence-specific probes Sequence-specific probes
Thermal cycler All real-time cyclers (e.g. LC, RG, ABI)
With or without ROX Available with ROX in master mix and with ROX as a separate vial

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Kit Handbooks
2
QuantiFast Probe RT-PCR Kit
QuantiFast Probe RT-PCR +ROX Vial Kit For fast, quantitative, real-time, one-step RT-PCR using sequence-specific probes
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配列特異的なプローブを用いた高速な1ステップ・リアルタイム定量RT-PCR
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