QuantiFast SYBR® Green RT-PCR Kit
For fast, one-step qRT-PCR using SYBR Green I for gene expression analysis
For fast, one-step qRT-PCR using SYBR Green I for gene expression analysis
Cat. No. / ID: 204156
The QuantiFast SYBR Green RT-PCR Kit delivers fast and specific quantification of RNA targets by real-time one-step RT-PCR using SYBR Green I detection. Q-bond technology and an optimized, ready to use 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 qRT-PCR buffer system ensures highly specific and sensitive real-time quantification of RNA targets. The QuantiFast SYBR Green RT-PCR Kit is also supplied with an optimized RT mix for efficient cDNA synthesis in only 10 minutes. 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 SYBR Green RT-PCR Kit delivers highly specific and sensitive results, outperforming other real-time RT-PCR kits used in fast cycling mode (see figure " Specific one-step RT-PCR"). RT-PCR run times are reduced by up to 60% (see figure " Significantly reduced PCR times"), allowing you to get results faster. You can also greatly increase your sample throughput or efficiently share a cycler with other users.
Results in one-step RT-PCR are comparable to those achieved in two-step RT-PCR (see table).
|Mean CT value|
|Amount of cDNA/RNA||QuantiFast SYBR Green PCR Kit (two-step RT-PCR)||QuantiFast SYBR Green RT-PCR Kit (one-step RT-PCR)|
QuantiFast SYBR Green RT-PCR Kit enables accurate quantification of targets over several log dilutions of template (see figure " Detection over 7 logs of template"). Even small differences in the amount of low-copy targets can be clearly distinguished.
The QuantiFast SYBR Green RT-PCR Kit delivers highly sensitive and specific results over a wide dynamic range on both standard and fast cyclers. The fluorescent dye SYBR Green I in the master mix enables the analysis of many different targets without having to synthesize target-specific labeled probes. A specially developed fast RT-PCR buffer contains the novel PCR additive Q-Bond, which significantly reduces denaturation, annealing, and extension times (see figure " Fast primer annealing"). A balanced combination of K+ and NH4+ ions in the PCR buffer promotes specific primer annealing, enabling 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. The optimized QuantiFast RT Mix enables cDNA synthesis in just 10 minutes.
|Component||Features and benefits||Benefits|
|HotStarTaq Plus DNA Polymerase||5 min activation at 95ºC||Set up of qPCR reactions at room temperature|
|QuantiFast SYBR Green 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|
|SYBR Green I dye||Yields a strong fluorescent signal upon binding to double-stranded DNA||Highly sensitive quantification|
|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||Special blend of reverse transcriptases with high affinity for RNA||RNA can be transcribed in just 10 minutes, even through complex secondary structures|
The QuantiFast SYBR Green RT-PCR Kit is a ready-to-use master mix that eliminates the need for optimization of reaction and cycling conditions. Simply add primers and template to the ready-to-use PCR master mix, and start the reaction. Follow the protocol in the handbook to get fast and reliable results on any real-time cycler.
For optimal results in real-time, one-step RT-PCR, we recommend using the QuantiFast SYBR Green RT-PCR Kit in combination with QuantiTect Primer Assays. They are bioinformatically validated primer sets for any gene from human, mouse, rat, and many other species. Assays can be easily ordered online at the GeneGlobe Web portal.
The QuantiFast SYBR Green RT-PCR Kit is for use in gene expression analysis of RNA targets. QuantiFast SYBR Green RT-PCR Kits are compatible with all available real-time cyclers, including 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 SYBR Green RT-PCR Kit, which has been specially developed for fast cycling on these instruments.
|Applications||SYBR Green-based, real-time, one-step RT-PCR|
|Single or multiplex||Single|
|Real-time or endpoint||Real-time|
|SYBR Green I or sequence-specific probes||SYBR Green I|
|Reaction type||Real-time and one-step RT-PCR|
|Thermal cycler||All real-time cyclers (e.g. LC, RG, ABI)|
|With or without ROX||With ROX|
No, high annealing specificity with the QuantiFast SYBR Green PCR Kits is maintained due to an intrinsic hot-start and the buffer composition.
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.
We have performed numerous tests comparing the performance of QuantiFast Kits and QuantiTect Kits with QuantiTect Primer Assays. Due to the optimized ion concentrations in the PCR buffers, both perform equally well with QuantiTect Primer Assays and do not require any adjustment of primer concentration.
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.
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".
Any DNA contamination will artificially inflate the SYBR Green signal, yielding skewed gene expression profiles and false-positive signals. The most common source of DNA contamination is from PCR products generated during previous experiments. Such contamination is most often due to the improper disposal of tubes, tips, and gels that previously came into contact with PCR products. Additionally, PCR products may also contaminate pipettors, racks, work pads, and commonly used reagents such as water and buffers. To minimize the risk of contaminating your experiment with extraneous DNA, the following steps should be taken:
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.
QuantiTect Primer Assays are primer pairs designed and bioinformatically validated specifically for real-time RT-PCR with SYBR Green detection. To find a primer assay for your target gene of interest, please visit our GeneGlobe data base.
For best results, we strongly recommend using QuantiTect Primer Assays in combination with QIAGEN's products for SYBR Green-based Real-Time PCR and RT-PCR.
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).
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.
Note that these Assays are guaranteed for use with the QuantiTect or QuantiFast chemistries only!
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.
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.
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.
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 each QuantiTect Primer Assay, we retrieve the sequence of the target gene from curated databases and exclude SNP regions from assay design.
We then use our proprietary algorithm to design assays that amplify RNA sequences only (i.e., at least one primer overlaps a splice site), provided that information on the position of splice sites is available. The assays are designed to provide optimal performance with QuantiFast and QuantiTect SYBR Green Kits.
After assay design, we validate randomly selected QuantiTect Primer Assays using real-time RT-PCR to check their compatibility with various real-time cyclers. Both two-step and one-step RT-PCR are carried out. Successful amplification of the target is indicated by the following factors: high sensitivity, high efficiency, high specificity (i.e., a single peak in melting curve analysis), and no primer–dimers in the no-template control (NTC).
To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.
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.
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.
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 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.
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:
The Q-Bond Molecule, present in the PCR Buffer of the QIAGEN Fast Cycling PCR Kit and the QuantiFast Kits, dramatically increases the binding affinity of DNA polymerase to single-stranded DNA, thereby facilitating the reduction of annealing time to just a few seconds. It is a non-protein PCR component. Unfortunately, all further information on this molecule is proprietary.
The activation time for HotStarTaq Plus DNA Polymerase used in the QuantiFast SYBR Green PCR Kits is longer than that for QuantiFast Probe PCR Kits. This is due to differences in buffer composition. Buffer components such as salts and additives influence the time required for enzyme activation.
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.
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.
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.
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.
The two-step cycling protocols of the QuantiFast SYBR Green PCR Kits allow a significant reduction in cycling time. It is more effective than reducing individual times for annealing and extension.
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.