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QuantiFast Probe RT-PCR Plus Kit

For fast, one-step qRT-PCR using sequence-specific probes, including genomic DNA removal

  • Efficient removal of gDNA for accurate gene expression analysis
  • Highly suited for analysis of short, fragmented RNA from FFPE samples
  • Sensitive detection of two targets simultaneously
  • Ready-to-use, real-time one-step PCR master mix
  • Fast universal protocol for all cyclers

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.

Cat No./ID: 204482
QuantiFast Probe RT-PCR Plus Kit (80)
$179.00
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For 80 x 25 µl reactions: 0.5 ml 2x QuantiFast Mix 1, 0.5 ml 2x QuantiFast Mix 2, 20 µl RT Mix, 210 µl ROX Dye Solution, 210 µl High-ROX Dye Solution, 1.9 ml RNase-Free Water
Cat No./ID: 204484
QuantiFast Probe RT-PCR Plus Kit (400)
$701.00
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For 400 x 25 µl reactions: 1.3 ml 2x QuantiFast Mix 1 (x 2), 1.3 ml 2x QuantiFast Mix 2 (x 2), 100 µl RT Mix, 210 µl ROX Dye Solution, 210 µl High-ROX Dye Solution, 1.9 ml RNase-Free Water
The QuantiFast Probe RT-PCR Plus 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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QuantiFast Probe RT-PCR Plus Kit procedure

With the QuantiFast Probe RT-PCR Plus Kit procedure, genomic DNA contamination is effectively eliminated through an optimized incubation step. After addition of the master mix for real-time RT-PCR, reverse transcription and, subsequently real-time PCR, take place in the same tube.

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Efficient genomic DNA removal for accurate gene expression analysis.
Total RNA was purified from human breast, liver, or kidney FFPE samples with the RNeasy FFPE Kit. Duplex, real-time RT-PCR was performed with (+RT) or without (–RT) the reverse transcription step for a range of different targets with different expression levels using QuantiFast Probe Assays (FAM labeled) and [A] the QuantiFast Probe RT-PCR Plus Kit or [B] an alternative kit for multiplex PCR which does not include an integrated gDNA removal step. All reactions were performed on the ABI StepOnePlus cycler. [A] High CT values in the –RT samples indicate no amplification and the absence of genomic DNA contamination. Therefore the CT values obtained in the +RT samples reflect reliable and accurate detection of RNA. Targets 12 (TUSC2) and 13 (EZH2) are not expressed. These results show that the QuantiFast Probe RT-PCR Plus Kit efficiently removes genomic DNA. [B] Similar CT values in both the –RT and +RT samples indicate that contaminating genomic DNA is being detected, making these results unreliable.
Performance
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").
Principle

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.

Components of QuantiFast Probe RT-PCR Plus Kit
Component Features Benefits
QuantiFast RT-Mix Special blend of reverse transcriptases with high affinity for RNA, even through complex secondary structures RNA can be transcribed in just 20 minutes, even through complex secondary structures
2x QuantiFast Mix 1
gDNA Wipeout Buffer Effective elimination of genomic DNA Detection of RNA only in the qRT-PCR reaction
2x QuantiFast Mix 2*
HotStarTaq Plus DNA Polymerase 5 min activation at 95ºC Set up of qPCR reactions at room temperature
QuantiFast Probe RT-PCR Plus Reagents Balanced combination of NH4+ and K+ ions Specific primer annealing ensures reliable PCR results
Synthetic Factor MP Reliable multiplexing analysis of up to 4 genes in the same tube
Unique Q-Bond additive Faster PCR run times, enabling faster results and more reactions per day
* Also contains a dNTP mix (dATP, dCTP, dGTP, and dTTP).
Procedure

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.

Applications

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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Brochures & Guides (1)
Critical factors for molecular analysis of FFPE samples
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FAQs (18)
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Kit Handbooks (5)
For quantitative, probe-based, real-time one-step RT-PCR with integrated genomic DNA removal
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For fast, real-time RT-PCR using sequence-specific, hydrolysis probes in combination with QuantiFast Kits
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英語版Handbookの記載内容の訂正
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ゲノムDNA 除去を組み込んだ、プローブ法によるリアルタイム定量1 ステップRT-PCR 用
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QuantiFast Kit と配列特異的な加水分解プローブを組み合わせた高速リアルタイムRT-PCR
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