QuantiFast Probe RT-PCR Plus Kit
For fast, one-step qRT-PCR using sequence-specific probes, including genomic DNA removal
The QuantiFast Probe RT-PCR Plus Kit is highly suited for gene expression analysis of RNA from all sources, including short, fragmented RNA (e.g., RNA from formalin-fixed, paraffin-embedded [FFPE] samples). This kit provides rapid, real-time one-step RT-PCR quantification of 1 or 2 RNA targets in a single tube using probe detection (e.g., using hydrolysis [TaqMan®] probes like the type provided with QuantiFast Probe Assays). In order to avoid false-positive signals, genomic DNA contamination is effectively eliminated through an optimized incubation step prior to real-time RT-PCR. ROX dye is provided in separate tubes at two different concentrations for fluorescence normalization, enabling use of the kit on any real-time cycler. For convenience, the master mix can be stored at 2–8°C.
The QuantiFast Probe RT-PCR Plus Kit provide fast and sensitive quantification, even in samples containing highly degraded RNA (see figure "Highly efficient real-time RT-PCR detection of degraded RNA"), which can contain high levels of residual genomic DNA (see figure "Efficient genomic DNA removal for accurate gene expression analysis").
The QuantiFast Probe RT-PCR Plus Kit delivers fast and highly sensitive quantification of RNA targets in a singleplex or duplex format using sequence-specific probes (e.g., hydrolysis [TaqMan®] probes provided with QuantiFast Probe Assays). To enable accurate measurement of RNA for gene expression analysis, genomic DNA contamination is effectively eliminated through a brief incubation step prior to real-time RT-PCR. The presence of genomic DNA can result in false positive RT-PCR signals with lower CT values than true positives.
Amplifying reference and target genes in the same reaction instead of in separate reactions increases the reliability of gene quantification by minimizing handling errors. The QuantiFast Probe RT-PCR Plus Kit delivers highly sensitive and rapid results over a wide dynamic range on both standard and fast cyclers without optimization (see flowchart "QIAGEN multiplex kits"). The specially developed fast PCR buffer contains the novel additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure "Fast primer annealing"). High specificity and sensitivity in one-step RT-PCR are achieved without any time-consuming optimization steps through the use of a specialized RT-PCR buffer, which contains a balanced combination of K+ and NH4+ ions to promote specific primer annealing, while the unique Factor MP stabilizes specifically bound primers (see figure "Unique PCR buffer"). In addition, HotStarTaq Plus DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products. The kit is also supplied with an optimized RT mix for efficient cDNA synthesis in only 20 minutes. Two vials of ROX at different concentrations are provided for fluorescence normalization enabling use of the kit on any available real-time cycler. Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.
The QuantiFast Probe RT-PCR Plus Kit includes ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. Follow the protocol in the handbook to get fast and reliable results on any real-time cycler (see flowchart "QuantiFast Probe RT-PCR Plus Kit procedure").
QuantiFast Probe Assays are predesigned, genomewide assays that use hydrolysis, probe-based detection. They are delivered with the QuantiFast Probe RT-PCR Plus Kit for guaranteed results in singleplex or duplex, one-step qRT-PCR.
The QuantiFast Probe RT-PCR Plus Kit is highly suited for gene expression analysis of analytes from all sources, including FFPE tissue samples and degraded RNA samples. The kit can be used on fast cyclers with rapid ramping rates as well as on standard cyclers, including instruments from QIAGEN, Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent.
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