QuantiFast Probe RT-PCR Kits
For fast, one-step qRT-PCR using sequence-specific probes for gene expression analysis
For fast, one-step qRT-PCR using sequence-specific probes for gene expression analysis
QuantiFast Probe RT-PCR Kits deliver fast and sensitive real-time one-step RT-PCR quantification of RNA targets using probe-based detection. Q-bond technology and an optimized 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 PCR buffer system in the ready-to-use master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization. The kits are also supplied with an optimized RT mix for efficient cDNA synthesis in only 10 minutes. Two kit formats are available: the QuantiFast Probe RT-PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiFast Probe RT-PCR +ROX Vial Kit for all other cyclers. For convenience, the master mix can be stored at 2–8°C.
IMPORTANT NOTE: As announced earlier, the production of the QuantiFast kits has been discontinued since mid-2021. Hence, these products will be 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.
QuantiFast Probe RT-PCR Kits deliver highly sensitive results, outperforming other real-time RT-PCR kits (see figure " Sensitive One-step RT-PCR"). RT-PCR run times are reduced by up to 60% (see figure " Significantly reduced PCR times"), allowing you to achieve fast PCR results without compromising on RT-PCR performance (see figure " Faster results without compromising sensitivity"). You can also greatly increase your sample throughput or efficiently share a cycler with other users. With QuantiFast Probe RT-PCR Kits, you follow the same procedure whether you work with a standard or fast cycler.
QuantiFast Probe RT-PCR Kits allow accurate quantification over a wide dynamic range (see figure " Comparable dynamic range"). Template dilutions of up to 6 logs can be reliably detected (see figure " Wide dynamic range and high sensitivity").
QuantiFast Probe RT-PCR Kits deliver highly sensitive and rapid results over a wide dynamic range on both standard and fast cyclers. The kits are designed for use with all types of sequence-specific probes, including hydrolysis probe detection (e.g., TaqMan and other dual-labeled probes) and FRET probes. The optimized QuantiFast RT Mix enables cDNA synthesis in just 10 minutes and a specially developed fast RT-PCR buffer contains the novel additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure " Fast primer annealing"). The RT-PCR buffer also contains a balanced combination of K+ and NH4+ ions, which promote specific primer annealing and enable 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.
|Component||Features and benefits||Benefits|
|HotStarTaq Plus DNA Polymerase||5 min activation at 95ºC||Set up of qPCR reactions at room temperature|
|QuantiFast Probe RT-PCR Buffer||Balanced combination of NH4+ and K+ ions||Specific primer annealing ensures reliable PCR results|
|Unique Q-Bond additive||Faster PCR run times, enabling faster results and more reactions per day|
|ROX dye†||Normalizes fluorescent signals on Applied Biosystems and, optionally, Agilent instruments||Precise quantification on cyclers that require ROX dye. Does not interfere with PCR on any real-time cycler|
|QuantiFast RT Mix||Separate solution added during PCR setup||Fast cDNA synthesis in just 10 minutes|
QuantiFast Probe RT-PCR Kits contain ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. Simply add template RNA, primers, and probe to the master mix and follow the protocol in the handbook to get fast and reliable results on any real-time cycler. Kits are available with or without ROX passive reference dye in the master mix, enabling use on virtually any real-time cycler (see table). Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.
|ROX dye||Kit||Compatible cyclers|
|Supplied in master mix||QuantiFast Probe RT-PCR Kit||All cyclers from Applied Biosystems except Applied Biosystems 7500|
|Supplied in separate tube||QuantiFast Probe RT-PCR +ROX Vial Kit||Applied Biosystems 7500 and cyclers from|
Bio-Rad, Cepheid, Eppendorf, QIAGEN, Roche, Agilent, and other suppliers
QuantiFast Probe RT-PCR Kits can be used for probe-based gene expression analysis of RNA targets on any real-time cycler. This includes 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 Probe RT-PCR Kit, which has been specially developed for fast cycling on these instruments.
|Applications||Probe-based, real-time RT-PCR|
|Single or multiplex||Single|
|Real-time or endpoint||Real-time|
|Thermal cycler||All real-time cyclers (e.g. LC, RG, ABI)|
|Reaction type||Real-time one-step RT-PCR|
|SYBR Green I or sequence-specific probes||Sequence-specific probes|
|With or without ROX||Available with ROX in master mix and with ROX as a separate vial|
Yes, QuantiFast Kits can also be run on a qPCR cycler without fast cycling options. You cannot achieve rapid ramping rates, but you can still take advantage of the combined annealing/extension step and the reduced denaturation and annealing/extension times offered by QuantiFast Kits.
You will be able to obtain your PCR results in a much shorter time.
QuantiFast Kits for 400 x 25 µl reactions contain a master mix that is aliquoted into 3 separate tubes.
QuantiFast Kits for 2000 x 25 µl reactions provide one tube containing 25 ml master mix to offer a cost-effective solution for higher throughput experiments.
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 : 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 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.
Yes, the QuantiFast SYBR Green PCR Kit and QuantiFast Probe PCR Kits are available for 80 x 25 µl reactions. This trial-kit size is not available for QuantiFast RT-PCR Kits.
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".
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.
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.
Compared with QuantiTect Kits, the recommended reaction volume for QuantiFast Kits is reduced from 50 µl to 25 µl (96-well block cyclers), and from 20 µl to 10 µl (384-well block cyclers).
The volume of master mix remains the same, which means that QuantiFast Kits offer twice the number of reactions as QuantiTect Kits. However, for LightCycler instruments, the recommended reaction volume remains the same (20 µl).
The combination of Omniscript and Sensiscript Reverse Transcriptases was optimized in the QuantiFast RT-PCR Kits. In addition, an optimized dNTP concentration and the limitation of amplicon size to <300 bp allow to reduce the time for the reverse transcription step to only 10 minutes.
Yes, please choose from the Supplementary Protocols below:
QuantiFast Probe RT-PCR +ROX Vial Kit:
The storage time for QuantiFast PCR Kits is shorter than for QuantiTect PCR Kits, because all QuantiFast master mixes contain HotStarTaq Plus DNA Polymerase, instead of HotStarTaq DNA Polymerase which requires longer activation times.
Excessive exposure to elevated temperatures will result in reactivation of the HotStarTaq Plus DNA Polymerase, eventually leading to nonspecific amplification.
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.
Our unique multiplex PCR buffer system with ammonium and potassium ions and Factor MP has been further optimized in QuantiFast and Rotor-Gene Kits. We have also discovered Q-Bond, a buffer component which supports the rapid formation of the polymerase–primer–template complex, leading to reduced annealing times.
No. Optimized thermal cycling programs for use with QuantiFast Kits and a Program Selection Guide are available.
To install these programs on your Mastercycler ep realplex, contact your Eppendorf sales representative or visit our QIAGEN/Eppendorf Alliance page.
No. We recommend using QuantiFast Probe +ROX Vial Kits: the master mix does not contain ROX dye, but the kit is supplied with a separate tube of ROX dye. The kits allow to adjust the ROX concentration in the master mix according to your cycler’s requirements.
Please follow specific recommendations in the QuantiFast Probe PCR Handbook.
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.
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.
We recommend a reaction volume of 10 µl when using 384-well blocks with QuantiFast PCR Kits. If reducing the reaction volume to 2 µl, results will vary depending on the real-time cycler used.
Please contact QIAGEN Technical Services for more information.
This is due to differences in composition between PCR and RT-PCR buffers. QuantiFast PCR Buffers are optimized for fast amplification with shortest possible PCR steps, while QuantiFast RT-PCR Buffers are optimized for reverse transcription and subsequent amplification.
Rotor-Gene Kits are specifically developed for the Rotor-Gene Q PCR Cycler. The unique rotary system of the cycler combined with the kits’ proprietary buffer system enable ultrafast cycling. Rotor-Gene Kits do not contain ROX dye since no normalization to a passive reference is required. Also, Rotor-Gene Kits do not contain dUTP; therefore, UNG pretreatment is not possible.
Yes, we offer Quantifast one-step RT-PCR kits for Probe and SYBR Green detection:
No. The master mix in QuantiFast PCR Kits contains only dTTP. To perform a UNG treatment, we recommend using QuantiTect Kits.
We have compared QuantiFast Kits and QuantiTect Kits using around 30 different assays (using both SYBR Green and Probe detection for each assay).
QuantiFast Kits gave identical or sometimes better Ct values than QuantiTect Kits (except for very long amplicons). Therefore, scientists switching from QuantiTect to QuantiFast Kits can, in most cases, obtain comparable results.
The master mix in QuantiFast SYBR Green Kits contains an optimized concentration of ROX dye that works well with all cyclers.
QuantiFast Probe PCR Kits are available in two formats:
We recommend using the latter with the Applied Biosystems 7500 Fast System. Use the ROX concentration indicated in the QuantiFast Probe PCR Kits handbook.
Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.
Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line. Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.
If the issue persists, please send the original run file to QIAGEN Technical Services.
No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.
Ensure PCR amplicons are as short as possible, ideally 60–150 bp. Always use the same algorithm or software to design the primers and probes. For optimal results, only combine assays that have been designed using the same parameters.
Check the functionality of each set of primers and probes in individual assays before combining the different sets in the multiplex assay. Choose compatible reporters and quenchers based on a specific instrument. See How do I select appropriate reporter and quencher combinations for multiplex PCR.
For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.
The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.
Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.
Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (Tm) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer Tm, but approximately 3ºC lower than the specific amplicon Tm.
Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:
For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.
The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.
If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.
Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.