QuantiFast Probe RT-PCR Plus Kit

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

Features

  • 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
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QuantiFast Probe RT-PCR Plus Kit (400)

Cat. No. / ID: 204484

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
This product will be discontinued as of June 30, 2022 or until stocks last. We recommend 208354.
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.

✓ 24/7 automatic processing of online orders

✓ Knowledgeable and professional Product & Technical Support

✓ Fast and reliable (re)-ordering

Product Details

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.

 

IMPORTANT NOTE: This product is phasing-out until June 30, 2021, and afterward, available only until stocks last. Visit the product page of the successor kit to view improved features or to request a trial kit.

 

For more information and FAQs on this transition, visit: www.qiagen.com/PCRresource.

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").
See figures

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).
See figures

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.

See figures

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.

Supporting data and figures

Resources

Brochures & Guides (2)
Sample to Insight solutions for successful molecular analysis
Critical factors for molecular analysis of FFPE samples
Kit Handbooks (2)
For fast, real-time RT-PCR using sequence-specific, hydrolysis probes in combination with QuantiFast Kits
For quantitative, probe-based, real-time one-step RT-PCR with integrated genomic DNA removal
Safety Data Sheets (1)
Download Safety Data Sheets for QIAGEN product components.
Additional Resources (1)
Quick-Start Protocols (1)

FAQ

How many freeze-thaw cycles can the master mix contained in the QuantiFast Probe RT-PCR Plus Kit tolerate?
The master mixes of the QuantiFast Probe RT-PCR Plus Kit can undergo up to 10 freeze-thaw cycles without any reduction in performance. For optimal long-term storage, aliquot the master mix, and store it at -20°C in sterile, polypropylene tubes.
FAQ ID -2357
What are important points regarding the genomic DNA wipeout buffer in the QuantiFast Probe RT-PCR Plus Kit?
  • The gDNA removal should be performed at room temperature from 15-30°C.
  • Perform the gDNA removal step for 5 minutes. However, the incubation can be prolonged to 15 minutes. An incubation longer than 15 minutes is not recommended.
  • The gDNA elimination reaction is efficiently stopped by adding the QuantiFast Mix 2.

 

 

 

FAQ ID -2359
I would like to order the QuantiFast Probe Assays without master mix. Is this possible?

QuantiFast Probe Assays are only sold in combination with optimized real-time RT-PCR master mixes and cannot be sold as stand-alone assays.

 

FAQ ID -2360
Do you have any information or guidelines regarding the choice of reference genes for real-time PCR?

Yes, please visit our website section 'Using endogenous control genes in real-time RT-PCR' for general information. It provides a list of relative gene expression levels for commonly used human and mouse reference genes.

We offer a set of ready-to-order control genes for use in SYBR Green based as well as probe based real-time RT-PCR.

In addition, you may want to refer to the following citations on reference gene selection for quantitative real-time PCR:

• Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, DePaepe A, Speleman F [2002]: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3:0034.

• Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A., 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 313(4): 856-62.

• Katrien Smits,Karen Goossens, Ann Van Soom, Jan Govaere, Maarten Hoogewijs, Emilie Vanhaesebrouck,Cesare Galli, Silvia Colleoni, Jo Vandesompele, and Luc Peelman [2009]Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts. BMC Res Notes. Dec 11;2:246.

FAQ ID -2371
How does the QuantiFast Probe RT-PCR Plus Kit eliminate genomic DNA contamination?
Genomic DNA contamination is effectively eliminated through an optimized incubation step prior to real-time RT-PCR step.
FAQ ID -2354
Why do replicates in real-time PCR have different plateau heights?

Replicates in real-time PCR may have different plateau heights due to differences in the reaction kinetics for each sample. Even though replicates start out with identical template amounts, the rate at which reagents are being depleted, and the point when exponential accumulation of PCR product stops and becomes linear, differ between replicates. This will result in different plateau heights, the stage where PCR reactions have come to a halt, and little or no additional PCR product is being amplified. You can find further information in Chapter 'Quantification of target amounts' of our Brochure "Critical Factors for Successful Real-Time PCR".

 

FAQ ID -539
How should fluorescent labeled probes be stored?

Fluorescent oligonucleotides should be stored in the dark, as light can slowly degrade the fluorescent moieties. For optimal long-term storage of fluorescent dye-labeled probes (except Cyanine 570, Cy3.5, Cyanine 670, and Cy5.5), the oligos should be resuspended in a slightly basic solution (e.g., TE buffer at pH 8.0). If resuspended below pH 7.0, the probe can degrade. We recommend to aliquot the sample, and store the aliquots at -20°C.

Note that Cyanine 570, Cy3.5, Cyanine 670, and Cy5.5 begin to degrade at a pH above pH 7.0. For best results, resuspend Cy-labeled oligos at pH 7.0, aliquot, lyophilize, and store at -20°C.

FAQ ID -784
Do you have information on the use of recombinant DNA and RNA as absolute standards for realtime RT-PCR?

Recombinant DNA (recDNA) is very stable and represents the average size of mRNA. Due to the cloning and purification processes, obtaining recDNA can lengthen the overall process of generating standards.

Recombinant RNA (recRNA) and native RNA undergo reverse transcription as well as PCR, and mimic the natural process for mRNA in RT-PCR. Complicated cloning and purification of recRNA and instability of recRNA are two disadvantages for using recRNA as a standard. For further details please refer to the section "Generating Standard Curves" in Appendix D of the QuantiTect SYBR Green PCR Handbook.

FAQ ID -729
What are the differences between the existing QuantiFast Probe RT-PCR Kit and the new Plus Kit?
QuantiFast Mix 1 is delivered with the QuantiFast Probe RT-PCR Plus kit which contains genomic DNA Wipeout Buffer to eliminate residual gDNA. The rest of the procedure is comparable to the existing QuantiFast Probe RT-PCR Kit.
FAQ ID -2358
Can I do singleplex PCR with the QuantiFast Probe Assays?
Yes, for 2-step RT-PCR, the QuantiFast Probe PCR Kit can be used, for 1-step RT-PCR, the QuantiFast Probe RT-PCR Plus Kit is combined with the assays.
FAQ ID -2362
How important is the RNA purification process, for obtaining reliable qRT-PCR results?

The most important prerequisite for any gene expression analysis experiment is the preparation of consistently high-quality RNA from every experimental sample. Contamination by DNA, protein, polysaccharide, or organic solvents can jeopardize the success of an experiment.

Genomic DNA contamination in an RNA sample compromises the quality of gene expression analysis results. The contaminating DNA inflates the OD reading of the RNA concentration. It is also a source of false positive signals in RT-PCR experiments.

RNase contamination degrades RNA samples whichcauses low signal and false-negative results in PCR.

Residual polysaccharides, collagen, other macromolecules, and organic solvents in an RNA sample can inhibit the activity of DNase, which may interfere with DNase treatment for genomic DNA removal. These contaminants may also inhibit reverse transcriptase and DNA polymerase, leading to lower reverse transcription efficiency and reduced PCR sensitivity.

For fast purification of high-quality RNA we recommend QIAGEN’s RNeasy Kits like the RNeasy Mini Kit, the RNeasy Plus Universal Kit, or the RNeasy FFPE Kit.

FAQ ID -2655
What is the threshold cycle or Ct value?
The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.
FAQ ID -2682
Why does my realtime PCR assay quality decrease over time?
Make sure that template, primers, probes, and amplification reagents are stored correctly and avoid multiple freeze–thaw cycles for oligonucleotides and template. Check the performance of your real-time instrument as some instruments require the halogen lamp to be frequently replaced. Lasers must also be replaced occasionally.
FAQ ID -589
What are the recommended storage conditions of the QuantiFast Probe RT-PCR Plus Kit components?
The QuantiFast Probe RT-PCR Plus kit should be stored immediately upon receipt at –20°C in a constant-temperature freezer and protected from light. The 2x QuantiFast Mix 2 (Probe) can also be stored protected from light at 2–8°C for up to 1 month without showing any reduction in performance.
FAQ ID -2355
Why should DNA or cDNA targets be less than 250 bp long for real-time PCR?

Shorter amplification products facilitate high PCR efficiencies. Ideally, amplicon length should be less than 150 bp for optimal amplification efficiency. PCR efficiencies close to 100% are a crucial prerequisite for accurate quantification of target copy numbers in real-time PCR.

FAQ ID-751
What should I use as a standard for absolute quantification in real-time PCR?

For quantification of RNA, we strongly recommend using RNA molecules as standards. Use of in vitro transcripts as standards takes into account the variable efficiency of the RT reaction. An alternative to the use of in vitro transcripts as RNA standards is the use of a defined RNA preparation (e.g., from a cell line or virus preparation), for which the absolute concentration of the target has already been determined.

For quantification of DNA, several types of DNA can be used, such as plasmids, PCR products, or genomic DNA.

For more information, see Appendix E 'Generating Standard Curves' in the QuantiTect Probe PCR Handbook.

FAQ ID -1085
Is it necessary to perform calibration steps for the use of the MAX dye in duplex RT-PCR experiments on different cyclers?

It is not necessary to perform calibration steps with MAX dye.  For instruments from Applied Biosystems, simply use the VIC channel/filter for the detection of MAX. The emission maxima of MAX and VIC are very similar (557nm and 554nm), respectively. On the Rotor-Gene Q, use the yellow channel. On the BioRad CFX, use channel 2 for MAX detection. On the LightCycler 480, use the combination 523 nm (Excitation) / 568 nm (Emission).  MAX was chosen as a second dye label because VIC label is protected by ABI and HEX showed more crosstalk in our experiments.

FAQ ID -2373
How can I adjust the ROX concentration in the QuantiFast Probe RT-PCR Plus master mix?
The master mix supplied with the QuantiFast Probe RT-PCR Plus Kit contains no ROX dye. In order to adjust the reaction for instruments from Applied Biosystems (models 7000, 7300, 7700, 7900HT, StepOne™, and StepOnePlus™ Real-Time PCR Systems), it is necessary to add High-ROX Dye Solution to the master mix. For Applied Biosystems 7500 and instruments from Stratagene, ROX dye is required at a lower concentration. This requires the user to add the supplied ROX Dye Solution to the master mix during reaction setup (see the kit handbook for details).
FAQ ID -2356