QuantiFast SYBR® Green RT-PCR Kit

For fast, one-step qRT-PCR using SYBR Green I 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
  • Universal protocol for all standard and fast cyclers
The QuantiFast SYBR Green RT-PCR Kit delivers fast and specific quantification of RNA targets by real-time one-step RT-PCR using SYBR Green I detection. Q-bond technology and an optimized, ready to use 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 qRT-PCR buffer system ensures highly specific and sensitive real-time quantification of RNA targets. The QuantiFast SYBR Green RT-PCR Kit is also supplied with an optimized RT mix for efficient cDNA synthesis in only 10 minutes. For convenience, the master mix can be stored at 2–8°C.
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QuantiFast SYBR Green RT-PCR Kit (400)
For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast SYBR Green RT-PCR Master Mix (contains ROX dye), 100 µl QuantiFast RT Mix, 2 x 2 ml RNase-Free Water
204154
977,00 €
QuantiFast SYBR Green RT-PCR Kit (2000)
For 2000 x 25 µl reactions: 25 ml 2x QuantiFast SYBR Green RT-PCR Master Mix (contains ROX dye), 0.5 ml QuantiFast RT Mix, 20 ml RNase-Free Water
204156
3.902,00 €
The QuantiFast SYBR® Green RT-PCR Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Significantly reduced RT-PCR times.|Specific one-step RT-PCR.|Detection over 7 logs of template.|Specific primer annealing.|Fast primer annealing.|
The QuantiFast SYBR® Green RT-PCR Kit reduces total RT-PCR run time by up to 60% in real-time one-step RT-PCR on standard cyclers (40 cycles without melting curve analysis; comparison with standard QIAGEN real-time PCR kits). L: LightCycler 2.0; A1: ABI PRISM 7900; A2: Applied Biosystems 7500; A3: ABI PRISM 7000.|Expression of BCL2 (an apoptosis gene) in HeLa cells was analyzed by one-step RT-PCR on the LightCycler 2.0. Duplicate reactions were run using 10-fold RNA dilutions (100 ng to 0.1 ng). Unlike [B] the instrument-dedicated kit from Supplier R (which was used according to the fast cycling protocol), the [A] QuantiFast SYBR® Green RT-PCR Kit provided specific amplification (single peak in melting curve analysis) and sensitive quantification (lower CT values that are evenly spaced from each other).|The QuantiFast SYBR® Green RT-PCR Kit provided accurate quantification of HSP89 (a heat shock protein) transcript over a wide dynamic range in a one-step RT-PCR reaction on the Applied Biosystems 7500 Fast System. Reactions were run in triplicate using 10-fold dilutions of in vitro transcript (107 down to 10 copies).|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

The QuantiFast SYBR Green RT-PCR Kit delivers highly specific and sensitive results, outperforming other real-time RT-PCR kits used in fast cycling mode (see figure "Specific one-step RT-PCR"). RT-PCR run times are reduced by up to 60% (see figure "Significantly reduced PCR times"), allowing you to get results faster. You can also greatly increase your sample throughput or efficiently share a cycler with other users.

Results in one-step RT-PCR are comparable to those achieved in two-step RT-PCR (see table).

Comparable performance in two-step and one-step RT-PCR on the Applied Biosystems 7500 Fast System
Mean CT value
Amount of cDNA/RNA QuantiFast SYBR Green PCR Kit (two-step RT-PCR) QuantiFast SYBR Green RT-PCR Kit (one-step RT-PCR)
10 ng 20.27 20.11
1 ng 23.44 23.46
0.1 ng 27.18 26.86
NTC 45.00 45.00
Similar CT values were achieved in two-step and one-step RT-PCR analysis of the expression of AK2 (an adenylate kinase) in T98G cells. Reactions were run in triplicate. NTC: no template control.

QuantiFast SYBR Green RT-PCR Kit enables accurate quantification of targets over several log dilutions of template (see figure "Detection over 7 logs of template"). Even small differences in the amount of low-copy targets can be clearly distinguished.

Principle

The QuantiFast SYBR Green RT-PCR Kit delivers highly sensitive and specific results over a wide dynamic range on both standard and fast cyclers. The fluorescent dye SYBR Green I in the master mix enables the analysis of many different targets without having to synthesize target-specific labeled probes. A specially developed fast RT-PCR buffer contains the novel PCR additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure "Fast primer annealing"). A balanced combination of K+ and NH4+ ions in the PCR buffer promotes specific primer annealing, enabling 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. The optimized QuantiFast RT Mix enables cDNA synthesis in just 10 minutes. 

Components of 2x QuantiFast SYBR Green RT-PCR Kit*
ComponentFeatures and benefits Benefits
HotStarTaq Plus DNA Polymerase 5 min activation at 95ºC Set up of qPCR reactions at room temperature
QuantiFast SYBR Green 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
SYBR Green I dye Yields a strong fluorescent signal upon binding to double-stranded DNA Highly sensitive quantification
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 10 minutes, even through complex secondary structures
* Also contains dNTP mix (dATP, dCTP, dGTP, dTTP).
Procedure

The QuantiFast SYBR Green RT-PCR Kit is a ready-to-use master mix that eliminates the need for optimization of reaction and cycling conditions. Simply add primers and template to the ready-to-use PCR master mix, and start the reaction. Follow the protocol in the handbook to get fast and reliable results on any real-time cycler.

For optimal results in real-time, one-step RT-PCR, we recommend using the QuantiFast SYBR Green RT-PCR Kit in combination with QuantiTect Primer Assays. They are bioinformatically validated primer sets for any gene from human, mouse, rat, and many other species. Assays can be easily ordered online at the GeneGlobe Web portal.

Applications

The QuantiFast SYBR Green RT-PCR Kit is for use in gene expression analysis of RNA targets. QuantiFast SYBR Green RT-PCR Kits are compatible with all available real-time cyclers, including 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 SYBR Green RT-PCR Kit, which has been specially developed for fast cycling on these instruments.

Feature
Specifications
Applications SYBR Green-based, real-time, one-step RT-PCR
Reaction type Real-time and 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 SYBR Green I
Thermal cycler All real-time cyclers (e.g. LC, RG, ABI)
With or without ROX With ROX

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Kit Handbooks
4
For fast, quantitative, real-time, one-step RT-PCR using SYBR Green I
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For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection
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ゲノムワイドで即使用可能なリアルタイムRT-PCRアッセイ(SYBR Green検出)用
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SYBR Green I 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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