Cat. No. / ID: 208252
Featuring enhanced specificity, sensitivity, speed, and process safety, the QuantiNova SYBR Green PCR Kit (SYBR qPCR kit) sets new standards in SYBR Green-based qPCR. A novel hot-start mechanism significantly increases the specificity of real-time PCR and optimized chemistry produces accurate quantitative results from cDNA or gDNA on any real-time PCR cycler with detection of even a single target copy. Finally, a built-in visual indicator ensures correct pipetting.
Avoid the deleterious effects of mispriming at lower temperatures with the QuantiNova Probe PCR Kit (probe qPCR kit). Based on a novel, antibody-mediated, hot-start mechanism, the kit enhances the specificity and efficiency of probe-based real-time PCR to provide accurate, singleplex or duplex, cDNA or gDNA analysis on various real-time PCR cyclers. The added convenience of extreme stability for up to 100 hours at room temperature, without a need for any cooling agent, makes it ideal for handling of high-throughput samples and automated workflows. An in-built tracking system for visual identification of correct pipetting gives you absolute peace of mind while extremely sensitive detection of even low target amounts ensures reliable qPCR results every time.
QuantiNova Multiplex PCR Kits (multiplex qPCR kits) enable fast and reliable quantification of up to 5 cDNA or gDNA targets in a single tube by multiplex, real-time PCR or two-step RT-PCR. Q-bond technology and an optimized master mix promote ultrafast multiplex real-time PCR within 1 hour. The combination of a unique hot start and PCR buffer system in the ready-to-use 4x master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization, also providing automated reaction set up options at room temperature. An in-built tracking system for visual identification of correct pipetting helps to prevent human errors, and the combination with QuantiNova Reverse Transcription Kit for cDNA synthesis allows inclusion of QuantiNova Internal Control RNA to monitor successful reverse transcription and qPCR.
Want to try the QuantiNova SYBR Green PCR Kit for the first time? Request a quote for a trial kit.
Want to try the QuantiNova Probe PCR Kit for the first time? Request a quote for a trial kit.
Want to try QuantiNova Multiplex PCR Kits for the first time? Request a quote for a trial kit
Built-in visual indicator for accurate reaction setup
The master mix supplied with QuantiNova PCR Kits contains an inert blue dye that does not interfere with the real-time PCR, but increases visibility in the tube or well. The QuantiNova Yellow Template Dilution Buffer contains an inert yellow dye. When template nucleic acid is diluted in QuantiNova Yellow Template Dilution Buffer and added to the master mix, the color of the solution changes from blue to green (see figure “ Accurate reaction setup indicated by the built-in pipetting control”), providing a visual indication that each reaction was set up correctly.
QuantiNova SYBR Green PCR Kit
The high sensitivity of the QuantiNova SYBR Green PCR Kit results in accurate and robust detection of even a single target copy (see figure “ Robust and sensitive detection of single-copy targets”). This extreme sensitivity is achieved even under ultrafast cycling conditions.
The QuantiNova SYBR Green PCR Kit can be used with a wide range of template amounts. The performance of this SYBR real-time PCR kit has been tested for a dynamic range spanning 8 orders of magnitude (from 100 ng to 10 fg cDNA) and regardless of instrument used, quantification was accurate and robust (see figure “ Accurate quantification over a wide dynamic range”).
The stringent specificity and exceptional sensitivity of the QuantiNova chemistry produces optimal results on the QIAGEN Rotor-Gene Q, but can be used on any real-time PCR cycler, regardless of format, fast-cycling capacity, and need for a passive reference dye. ROX provided with the QuantiNova SYBR Green PCR Kit is simply added to the master mix if required. Amplification and quantification results using the Rotor-Gene Q, Agilent Technologies Mx3005P, Applied Biosystems 7900 HT Fast, ViiA 7, StepOnePlus, and 7500 Fast, Roche LightCycler 480, and Bio-Rad CFX96 are invariably robust and sensitive.
The QuantiNova SYBR Green PCR Kit delivers superior results compared to other SYBR Green PCR kits. Regardless of cycling conditions or instrument, the QuantiNova chemistry demonstrably generates lower CT values, higher reproducibility, and higher reaction efficiency.
The QuantiNova Yellow Template Dilute Buffer provides peace of mind during reaction setup but is not required to obtain superior results with the QuantiNova SYBR Green PCR Kit. The QuantiNova chemistry is designed to be flexible, so it can be incorporated into any established workflow. Thus, performance is optimal with your amplification target of interest, and your instrument and dilution buffer of choice.
QuantiNova Probe PCR Kit
The real-time PCR mix of this probe real-time PCR kit can be stored at 30°C for up to 100 hours without impairing the performance of subsequent reactions. The outstanding stability, even after extended storage at room temperature without the use of any cooling agent, makes the QuantiNova Probe PCR Kit ideal for high-throughput reaction setup and plate-stack handling (see table "Effect of storage on reaction stability".
|Mean CT values|
|QuantiNova Probe PCR Kit||Probe PCR kit from supplier L|
|Amount in ng||0 h||100 h||0 h||100 h|
|No template control||Not detected||Not detected||Not detected||Not detected|
Comparison of CT values before and after 100 hours storage at 30°C. Real-time PCRs including master mix, template cDNA, primers, and probes for TNF were incubated at 30°C for 100 hours and run in the real-time PCR together with freshly prepared reactions. Triplicate reactions of 3 different template amounts were tested. In contrast to the probe PCR kit from supplier L, the CT values provided by the QuantiNova Probe PCR Kit did not vary after extended storage of the samples at 30°C.
The high sensitivity of the QuantiNova Probe PCR Kit results in accurate and robust detection of even a single target copy (see figure “ Sensitive and robust detection of a single target copy”). The special master mix supplied with these kits enables accurate quantification of 2 targets having widely differing abundance in a single tube. This saves time, money, and reduces the amount of sample material needed. Moreover, the duplex PCR data obtained is comparable with that obtained from a singleplex PCR.
The unique combination of QIAGEN's proprietary and well-proven buffer technology along with the new QuantiNova DNA Polymerase, QuantiNova Antibody, and QuantiNova Guard, ensures real-time PCR success at the first attempt, without the need for costly and time-consuming optimization, even with challenging real-time PCR assays (see figure “ Superior results with challenging assays”).
The QuantiNova Probe PCR Kit can be used on any real-time cycler. ROX is provided in a separate tube and can be added if using a cycler that uses ROX as a passive reference dye. The kit delivers highly consistent results among different cyclers in terms of sensitivity, reproducibility, and efficiency. The consistency of the results is maintained, despite varying fast-cycling capacities of the different cyclers that result in different cycling protocols.
QuantiNova Multiplex PCR Kits
The special master mix supplied with QuantiNova Multiplex PCR Kits allows rapid setup of multiplex reactions and delivers successful results at the first attempt, providing multiplex PCR data that are comparable with singleplex PCR data. The highly concentrated 4x master mix accommodates up to 800 ng template input ensuring outstanding sensitivity, even in up to 5plex reactions. The kit can clearly distinguish between small differences in the amount of template and provides accurate quantification of targets of widely differing abundance.
A novel, antibody-mediated, hot-start mechanism ensures outstanding specificity and allows reaction setup at room temperature, ideally suited for automated procedures. The specially developed fast PCR buffer contains the additive Q-Bond, which significantly reduces annealing and extension times, allowing multiplex qPCR in less than one hour. The visual pipetting control helps to prevent human errors and increases process safety, particularly if combined with the Internal Control RNA provided in the QuantiNova Reverse Transcription Kit for quantitative 2-step RT-PCR.
The QuantiNova Multiplex PCR Kit increases workflow efficiency by generating more insight from limited sample material by using ultrafast and in-process controlled multiplex qPCR.
QuantiNova PCR Kits deliver cDNA or gDNA analysis with the highest specificity because of a novel, antibody-mediated, hot-start mechanism (see figure “ Principle of the novel QuantiNova hot-start mechanism”). At low temperatures, QuantiNova DNA Polymerase is kept in an inactive state by the QuantiNova Antibody and the novel additive QuantiNova Guard, that stabilizes the complex. This improves the stringency of the hot-start and prevents extension of nonspecifically annealed primers and primer dimers. Within 2 minutes of raising the temperature to 95°C, the QuantiNova Antibody and QuantiNova Guard are denatured and the QuantiNova DNA Polymerase is activated, enabling amplification.
Additionally, the QuantiNova chemistry includes features that streamline your workflow without loss of PCR sensitivity and efficiency. Inert dyes in the master mix and in the QuantiNova Yellow Dilution Buffer serve as a visual indicator that each reaction has been set up correctly. The blue color of the master mix increases visibility of contents in the reaction vessel and turns green upon adding template nucleic acids diluted in the QuantiNova Yellow Dilution Buffer (see figure “ Accurate reaction setup indicated by the built-in pipetting control”). The unique formulation of the PCR buffer included in QuantiNova SYBR PCR Kits enhances the stringency of the hot-start mechanism and shortens cycling steps so your PCR is done faster and your throughput can be increased.
QuantiNova Multiplex PCR Kits deliver highly sensitive and rapid results over a wide dynamic range on both standard and fast cyclers without optimization. The specially developed fast PCR buffer contains the additive Q-Bond, which significantly reduces annealing and extension times (see figure " Fast primer annealing").
Amplifying reference and target genes in the same reaction instead of in separate reactions increases the reliability of gene quantification by minimizing handling errors. Additionally the Internal Control RNA provided in the QuantiNova Reverse Transcription Kit for quantitative 2-step RT-PCR can be incorporated to monitor successful reverse transcription and qPCR.
QuantiNova Multiplex PCR Buffer includes a balanced combination of K+ and NH4+ ions as well as the unique synthetic Factor MP, which together promote stable and efficient annealing of primers and probes to the nucleic acid template, enabling high PCR efficiency (see figure " Unique multiplex PCR buffer promotes stable and efficient annealing").
The QuantiNova Multiplex PCR Kit master mix can conveniently be stored at 2–8°C for up to 12 months, and also the reaction set up is extraordinary stable at room temperature, allowing automated procedures to increase efficiency and accuracy.
QuantiNova PCR Kits contain ready-to-use master mixes that eliminates the need for optimization of reaction and cycling conditions. Simply add template gDNA or cDNA and primers (SYBR Green-based detection) or template gDNA or cDNA, primers and probes (probe-based detection) to the master mix and follow the protocol in the handbook to achieve rapid and reliable results on any real-time cycler. ROX passive reference dye is provided in a separate tube and the concentrations of the dye can be adjusted depending on the type of cycler used. Thus, QuantiNova PCR Kits can be used on virtually any real-time cycler, regardless of format or cycling conditions. Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.
QuantiNova Multiplex PCR Kits contain ready-to-use 4x master mixes that eliminate the need for optimization of reaction and cycling conditions. Simply add up to 800 ng template DNA and primer-probe sets to the master mix and follow the protocol in the handbook to get fast and reliable results on any real-time cycler. Kits provide ROX passive reference dye in a separate tube, to adjust appropriate ROX concentration, if required for your instrument.
For optimal results in real-time two-step RT-PCR, we recommend synthesizing cDNA using the QuantiNova Reverse Transcription Kit, which provides fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination. It additionally provides the QuantiNova Internal Control RNA, offering an in-process monitoring of successful reverse transcription and qPCR.
The QuantiNova SYBR Green PCR Kit can be used for SYBR Green-based gene expression analysis of cDNA targets and quantitative gDNA analysis on any real-time cycler. This includes instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, LIFE Technologies, Roche, and Agilent, as well as the Rotor-Gene Q.
QuantiNova Probe PCR Kits can be used for probe-based gene expression analysis of cDNA targets and quantitative gDNA analysis on any real-time cycler. This includes instruments from Applied Biosystems, Bio-Rad, Cepheid, Eppendorf, Roche, and Agilent.
QuantiNova Multiplex PCR Kits can be used for multiplex gene expression analysis of cDNA or gDNA targets on any real-time cycler. To fully exploit the capability of multiplex benefits, we recommend instruments providing up to 5plex capacity, such as the Rotor-Gene Q.
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.
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.
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.
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.
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:
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.