QuantiFast SYBR® Green RT-PCR Kit
For fast, one-step qRT-PCR using SYBR Green I for gene expression analysis
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.
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).
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.
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.
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