QuantiFast Probe RT-PCR Plus Kit
For fast, one-step qRT-PCR using sequence-specific probes, including genomic DNA removal
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.
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.
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.
|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|
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.
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.
QuantiFast Probe Assays are only sold in combination with optimized real-time RT-PCR master mixes and cannot be sold as stand-alone assays.
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.
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.
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.
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.
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.
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.