QuantiTect RT-PCR Kit

In one-step RT-PCR, both reverse transcription and amplification are performed in the same tube. Upon completion of reverse transcription, the reaction temperature is raised to reach denaturation/PCR enzyme activation temperature and the thermal cycling (PCR) begins. One-step RT-PCR generally uses gene-specific primers for both the RT and PCR steps. The procedure is fast, easy to automate, and minimizes the risk of contamination due to fewer handling steps.

In two-step RT-PCR, the RNA is first transcribed into cDNA using oligo-dT primers, random oligos, or gene-specific primers. An aliquot of the RT reaction is subsequently added to the real-time PCR reaction in a second tube. Choice of different types of RT primers allows the analysis of different transcripts by PCR from one RT reaction. Most commonly, an oligo-dT primer is used for the RT step, followed by PCR with a pair of gene-specific primers. Precious RNA samples can be immediately transcribed into more stable cDNA for later use and long-term storage.

The advantages of each method are summarized below:

Optimized one-step and two-step RT-PCR kits compatible with any real-time cycler are available from QIAGEN. For further details, please see our online section on 'Critical factors for successful gene expression assays', or download our Brochure 'Critical Factors for Successful Real-Time PCR'.

No. Uracil-N-Glycosylase will remain active during the reverse-transcription step with the QuantiTect Probe RT-PCR Kit, and destroy any newly synthesized cDNA. Heat-labile UNG is required for one-step RT-PCR. See also FAQ 564 for further info.

The reduced reaction volume recommended for QuantiFast PCR Kits compared to QuantiTect PCR Kits allows more efficient temperature transfer during short cycling steps.

The presence of ROX as passive reference dye in the Master Mix of the QuantiTect Multiplex PCR Kit and the QuantiFast Mutliplex PCR Kit limits the use of these kits to triplex PCR on 4-channel real-time cyclers. This is because the ROX dye occupies the channel for detecting probes labeled with ROX, Texas Red, or other equivalent dyes.

However, 4-plex PCR is possible on on instruments equipped with at least 5 channels. Alternatively, if you have a 4-channel real-time cycler that does not use ROX passive reference dye (e.g., iCycler iQ, Rotor-Gene 3000, Mx4000, Mx3000P, Smart Cycler II, LightCycler 2.0), you can use the QuantiTect Multiplex PCR NoROX Kit or the QuantiFast Multiplex PCR +R Kit to perform 4-plex PCR.

Please see table “Real-Time, Multiplex PCR on a Wide Range of Real-Time Cyclers” for compabilities of the QuantiTect Multiplex kits with different real-time cyclers.

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.

No. The master mixes in Rotor-Gene Kits contain dTTP instead of dUTP. If UNG treatment is required, we recommend using QuantiTect +UNG Kits. QuantiTect Kits are also compatible with the Rotor-Gene Q; however, the kits require a significantly longer cycling time.

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:

• Remove a single aliquot of water from your PCR-grade stock, sufficient to complete the experiment. This minimizes the number of times that the stock container is opened, thereby minimizing contamination risks.
• Use only fresh PCR-grade reagents and disposable labware.
• Treat any labware (tubes, tips, and tip boxes) used in PCR with 10% bleach, before discarding.
• Maintain a dedicated workspace for PCR setup (perhaps a PCR-only hood), away from areas of the lab where post-PCR work is done, such as running gels, enzyme digestions, and cloning.
• Change the lab bench pads/papers often and decontaminate lab benches and labware (racks, pipettors, etc.) before each use by washing with 10% bleach, and/or exposing to UV light for at least 10 minutes. This serves to degrade and/or inactivate contaminating DNA.
• Before, during, and after the experiment, minimize the opening and closing of any tubes or plates used during the experiment.  

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 primers of the QuantiTect Primer Assays are at a proprietary concentration that was specially optimized for sensitive and efficient amplification in any real time cycler. Always dilute these primers to a final work solution of 1x in your reaction, using either the QuantiTect SYBR Green PCR Kit or the QuantiTect SYBR Green RT-PCR Kit. Follow the instructions for use of the Primer Assays in the QuantiTect Primer Assay handbook.

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.

No, please do not exchange Quantiscript Reverse Transcriptase of the QuantiTect Reverse Transcription Kit with QuantiTect RT Mix of the QuantiTect Probe RT-PCR Kit.

Although both are an optimized mixture of Omniscript and Sensiscript Reverse Transcriptases, the mixture provided in the QuantiTect Reverse Transription Kit is optimized for random priming in a two-step reaction, whereas the mixture in the QuantiTect Probe RT-PCR Kit is optimized for gene-specific priming in a one-step RT-PCR reaction.

The annealing temperature for QuantiTect Primer Assays should be 55^oC when used with the QuantiTect SYBR Green PCR Kit and the QuantiTect SYBR Green RT-PCR Kit.

Use of the QuantiFast SYBR Green PCR and QuantiFast SYBR Green RT-PCR Kits requires a combined annealing/extension step at 60^oC, as described in the QuantiTect Primer Assay Handbook.

Note that these Assays are guaranteed for use with the QuantiTect or QuantiFast chemistries only!

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.

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.

Yes, QuantiTect Primer Assays work very well under fast-cycling conditions. Results achieved with QuantiFast SYBR Green Kits are comparable to those achieved with QuantiTect SYBR Green Kits.

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.

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.

QuantiTect Primer Assays are optimized for an annealing temperature of 55ºC when using the QuantiTect SYBR Green PCR Kit (for two-step RT-PCR) or the QuantiTect SYBR Green RT-PCR Kit (for one-step RT-PCR) .

Increasing the annealing temperature will reduce PCR performance (sensitivity and PCR efficiency) when using QuantiTect Primer Assays with these kits.

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.

We highly recommend any QuantiTect or QuantiFast Kit for quantitative PCR on cDNA generated with the QuantiTect Whole Transcriptome Kit.

Depending on the qPCR instrument, time savings when switching from standard cycling (e.g., QuantiTect PCR Kits) to fast cycling using QuantiFast Kits range from 40% to 60%.

Even when detecting low-abundance targets, we recommend using no more than 500 ng/reaction of DNA (for PCR kits) or RNA (for RT-PCR kits). Generally, 1–100 ng template should be sufficient and for abundant transcripts as little as 1 pg can be used.

Template purity is important if large volumes of low concentration template are to be added to the reaction. Using DNA or RNA purified with QIAGEN products, the template can contribute up to 40% of the final reaction volume as long as the recommended template amounts are not exceeded. If cDNA from an RT reaction is used as template, the volume of undiluted RT reaction added should not exceed 10% of the final PCR volume.

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:

• Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
• DNA template concentrations should be normalized using the same dilution buffer. Ensure the C_T values are below 30 and differ no more than 3 C_T values across individual samples.
• Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
• Always start with 0.7 µM primer concentration

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

No, QuantiTect Primer Assays are supplied as lyophilized, premixed primer pairs. Reaction components for SYBR Green real-time RT-PCR must be purchased separately.

• For two-step RT-PCR, the QuantiTect Reverse Transcription Kit and QuantiTect SYBR Green PCR Kit are recommended
• For one-step RT-PCR, the QuantiTect SYBR Green RT-PCR Kit is recommended

To find a QuantiTect Primer Assay for your target gene of interest, please visit our GeneGlobe data base.

Compatible reporter dye combinations for different real-time cyclers are listed in the "Important Notes" section under 'Suitable combinations of reporter dyes' in the QuantiTect Multiplex PCR Handbook and the QuantiTect Multiplex PCR NoROX 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.

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, probes and primers can be added to the QuantiTect Probe PCR- and the QuantiTect Probe RT-PCR Master Mix. This master mix can be stored at -20°C for up to 50 days, with a maximum of six freeze-thaw cycles (tested in our labs).

The master mix must be stored protected from light to avoid bleaching of the fluorescent probe. However, the QuantiTect RT Mix should be stored separately at –20°C, because the reverse transcriptases are sensitive to freezing in an aqueous environment. The QuantiTect RT Mix should be added to this master mix just prior to adding the template RNA and placing the reactions into the thermal cycler.

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.

Yes - dTTP in the QuantiTect Master Mix in QuantiTect SYBR Green and Probe PCR Kits is partially substituted by dUTP, therefore allowing treatment with uracil-N-glycosylase.

However, only heat-labile UNG can be used for one-step RT-PCR with the QuantiTect Probe RT-PCR and SYBR Green RT-PCR Kits. This is because heat stable UNG (isolated from E. coli) will destroy any cDNA synthesized during the RT step at 50°C. Heat-labile UNG will have lost any activity after a few minutes of the RT-step and therefore cannot interfere with cDNA synthesis.

For heat-stable uracil-N-glycosylase, please use the QuantiTect SYBR Green PCR +UNG (cat. no. 204163) or QuantiTect Probe PCR +UNG Kits (cat. no. 204363). Both kits include heat-stabile uracil-N-glycosylase. Although QIAGEN UNG is inactivated by incubation at 95°C for 15 minutes, the enzyme may exhibit residual activity at lower temperatures due to refolding. Therefore, it is recommended to perform subsequent PCR using a temperature of 55°C or above for the annealing step.

• LightCycler: We do not recommend performing real-time, multiplex PCR using TaqMan® probes or QuantiTect Assays on the LightCycler system due to the limitations of its optical detection system.
• LightCycler 2.0: Information on using the QuantiTect Multiplex PCR NoROX Kit with the LightCycler 2.0 system is provided in the QuantiTect Multiplex PCR NoROX Handbook. You will find the recommendation for the LightCycler 2.0 system in the chapter 'Important Notes' under "Suitable combinations of reporter dyes".

See trademarks.

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 (T_m) 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 T_m, but approximately 3ºC lower than the specific amplicon T_m.