Real-Time PCR Enzymes & KitsProbe-Based One-Step qRT-PCR12Probe-Based qPCR13Reverse Transcription & cDNA Synthesis for qPCR8SYBR Green- or Dye-Based One-Step qRT-PCR6SYBR Green- or Dye-Based qPCR10QuantiFast Probe RT-PCR Plus KitPrint For fast, one-step qRT-PCR using sequence-specific probes, including genomic DNA removal Efficient removal of gDNA for accurate gene expression analysisHighly suited for analysis of short, fragmented RNA from FFPE samplesSensitive detection of two targets simultaneouslyReady-to-use, real-time one-step PCR master mixFast universal protocol for all cyclersThe 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.Buy ProductsProduct DetailsProduct ResourcesBuy ProductsCat No./ID:204484QuantiFast Probe RT-PCR Plus Kit (400)$713.00Order ProductFor 400 x 25 µl reactions: 1.3 ml 2x QuantiFast Mix 1 (x2), 1.3 ml 2x QuantiFast Mix 2 (x 2), 100 µl RT Mix, 210µl ROX Dye Solution, 210 µl High-ROX Dye Solution, 1.9ml RNase-Free WaterThe 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.See trademarks.Product Details Fast primer annealing.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.Highly efficient real-time RT-PCR detection of degraded RNA.Total RNA (10 ng to 100 pg) from HeLa cells with a RIN value of 4.7 was amplified and detected in one-step RT-PCR on the ABI 7500 Fast cycler using [A] a QuantiFast Probe Assay for the human CENPM gene in combination with the QuantiFast Probe RT-PCR Plus Kit (amplicon size: 76 bp) or [B] a predesigned assay and kit from Supplier AII (amplicon size: 111 bp). Results from QuantiFast Reagents showed lower CT values and higher sensitivity compared to results using reagents from Supplier AII.QIAGEN multiplex kits.Unique PCR buffer.QuantiFast Probe RT-PCR Plus Kit procedureWith 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.PerformanceThe 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 cancontainhighlevels of residual genomic DNA (see figure "Efficient genomic DNA removal for accurate gene expression analysis").PrincipleThe 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 containsthe novel additive Q-Bond, whichsignificantly 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 ofa 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 isalso 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 kiton 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. Product ResourcesYou are not authorized to download the resourceFAQs (18) Sort optionsSort alphabetically What should I use as a standard for absolute quantification in real-time PCR?FAQ ID -1085ViewHow does the QuantiFast Probe RT-PCR Plus Kit eliminate genomic DNA contamination?FAQ ID -2354ViewWhat are the recommended storage conditions of the QuantiFast Probe RT-PCR Plus Kit components?FAQ ID -2355ViewHow can I adjust the ROX concentration in the QuantiFast Probe RT-PCR Plus master mix?FAQ ID -2356ViewHow many freeze-thaw cycles can the master mix contained in the QuantiFast Probe RT-PCR Plus Kit tolerate?FAQ ID -2357ViewWhat are the differences between the existing QuantiFast Probe RT-PCR Kit and the new Plus Kit?FAQ ID -2358ViewWhat are important points regarding the genomic DNA wipeout buffer in the QuantiFast Probe RT-PCR Plus Kit?FAQ ID -2359ViewI would like to order the QuantiFast Probe Assays without master mix. Is this possible?FAQ ID -2360ViewCan I do singleplex PCR with the QuantiFast Probe Assays?FAQ ID -2362ViewDo you have any information or guidelines regarding the choice of reference genes for real-time PCR?FAQ ID -2371ViewIs it necessary to perform calibration steps for the use of the MAX dye in duplex RT-PCR experiments on different cyclers?FAQ ID -2373ViewHow important is the RNA purification process, for obtaining reliable qRT-PCR results? FAQ ID -2655ViewWhat is the threshold cycle or Ct value? FAQ ID -2682ViewWhy do replicates in real-time PCR have different plateau heights?FAQ ID -539ViewWhy does my realtime PCR assay quality decrease over time?FAQ ID -589ViewDo you have information on the use of recombinant DNA and RNA as absolute standards for realtime RT-PCR?FAQ ID -729ViewWhy should DNA or cDNA targets be less than 250 bp long for real-time PCR?FAQ ID-751ViewHow should fluorescent labeled probes be stored?FAQ ID -784ViewShow allShow lessKit Handbooks (2) Sort optionsSort alphabetically (EN) - QuantiFast Probe RT-PCR Plus HandbookFor quantitative, probe-based, real-time one-step RT-PCR with integrated genomic DNA removalShow details(EN) - QuantiFast Probe Assay HandbookFor fast, real-time RT-PCR using sequence-specific, hydrolysis probes in combination with QuantiFast KitsShow detailsShow allShow lessAdditional Resources (1) Sort optionsSort alphabetically (EN) - Unlock the potential of your FFPE archiveShow detailsShow allShow lessQuick-Start Protocols (1) Sort optionsSort alphabetically QuantiFast Probe RT-PCR Plus Kit (EN)Show detailsShow allShow lessBrochures & Guides (2) Sort optionsSort alphabetically Discover the Real Potential of FFPE SamplesSample to Insight solutions for successful molecular analysisShow detailsSuccessful Biomarker Profiling from FFPE SamplesCritical factors for molecular analysis of FFPE samplesShow detailsShow allShow lessSafety Data Sheets (1) Sort optionsSort alphabetically MSDS QuantiFast Probe RT-PCR Plus Kit (400)Show allShow lessfragment fix placeholderCustomers who bought these products also boughtCat No./ID:211033QuantiTect Virus +ROX Vial Kit (200)For 200 x 50 µl reactions: QuantiTect Virus NR Master Mix (without ROX dye), ROX Dye Solution, QuantiTect Virus RT Mix, RNase-Free Water, QuantiTect Nucleic Acid Dilution Buffer$967.00Add To CartCat No./ID:9238279PAXgene 96 Incubator BlockBlock for denaturation of eluates in PAXgene 96 procedures$349.00Add To CartCat No./ID:208354QuantiNova Probe RT-PCR Kit (500)For 500 x 20 µl reactions: 3 x 1.7 ml QuantiNova Probe RT-PCR Master Mix, 100 µl QuantiNova Probe RT Mix, 100 µl Internal Control RNA, 500 µl Yellow Template Dilution Buffer, 1 ml ROX Reference Dye, 2 x 1.9 µl RNase-Free Water$683.00Add To CartCat No./ID:74116RNeasy Mini QIAcube Kit (240)For 240 RNA minipreps: RNeasy Mini Rotor Adapters (preloaded with spin columns and elution tubes); RNase-free reagents and buffers$1,621.00Add To CartCat No./ID:79256RNase-Free DNase Set (250)5 x 1500 Kunitz units RNase-free DNase I, RNase-free Buffer RDD, and RNase-free water for 250 RNA minipreps$528.00Add To CartCat No./ID:204354QuantiFast Probe PCR +ROX Vial Kit (400)For 400 x 25 µl reactions: 3 x 1.7 ml 2x QuantiFast Probe PCR Master Mix (without ROX dye), 210 µl ROX Dye Solution, 2 x 2 ml RNase-Free Water$526.00Add To CartCat No./ID:762635QIAsymphony PAXgene Blood RNA Kit (96)For 96 preps: 2 Reagent Cartridges, Enzyme Racks, accessories, and RNase-free buffers$1,134.00Add To CartCat No./ID:762164PAXgene Blood RNA Kit (50)50 PAXgene Spin Columns, 50 PAXgene Shredder Spin Columns, Processing Tubes, RNase-Free DNase I, RNase-Free Reagents and Buffers. 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