QuantiTect RT-PCR Kits

For one-step qRT-PCR using SYBR® Green I and one-step qRT-PCR and multiplex qRT-PCR using sequence-specific probes for gene expression analysis

Features

  • Highly sensitive detection of low-copy targets
  • qRT-PCR using sequence-specific probes or SYBR Green
  • Accurate quantification over several logs of template
  • No need to optimize reaction and cycling conditions
  • Detection of reference gene and up to 3 targets in the same tube
QuantiTect Probe RT-PCR Kit (200)

Cat. No. / ID: 204443

For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Probe RT-PCR Master Mix, 100 µl QuantiTect RT Mix, 2 x 2 ml RNase-Free Water
$692.00
Detection type
Probe
SYBR Green
Multiplex Probe
Size
QuantiTect Probe RT-PCR Kit (200)
QuantiTect Probe RT-PCR Kit (1000)
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QuantiTect RT-PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.

Product Details

QuantiTect RT-PCR Kits enable sensitive quantification of RNA targets by real-time one-step PCR using sequence-specific probes or SYBR Green I detection. The kits also allow reliable quantification of up to 5 RNA targets in a single tube by multiplex, real-time one-step RT-PCR. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qRT-PCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. For convenience, the master mixes in QuantiTect one-step RT-PCR kits can be stored at 2–8°C.

Two kit formats are available for multiplex RT-PCR using sequence-specific probes: the QuantiTect Multiplex RT-PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiTect Multiplex RT-PCR NoROX Kit for all other cyclers. The QuantiTect SYBR Green RT-PCR Kit is supplied with an optimized RT mix for efficient and sensitive reverse transcription over a wide range of RNA template amounts.

Performance

HotStarTaq DNA Polymerase included in QuantiTect one-step RT-PCR kits increases the specificity of the PCR reaction by providing the most stringent hot start compared with other polymerases.
The QuantiTect SYBR Green RT-PCR Kit contains a unique blend of reverse transcriptases, which delivers efficient and sensitive cDNA synthesis over a wide range of amounts of RNA template and enables specific quantification over a wide linear range (see figure " Comparable results in one-step and two-step RT-PCR").


The QuantiTect Probe RT-PCR Kit has a unique RT-PCR buffer that promotes highly specific annealing of primers and probes to the PCR template. HotStarTaq DNA Polymerase and the unique composition of the RT-PCR buffer enable the QuantiTect Probe RT-PCR Kit to provide sensitive quantification of low-copy RNA targets, as well as accurate quantification over a wide linear range (see figure " High sensitivity and efficiency, and wide dynamic range").

Performing reverse transcription and PCR sequentially in the same tube does not impair sensitivity, as demonstrated by CT values that are comparable to those achieved in real-time two-step RT-PCR (see figures " One-step RT-PCR with comparable performance to two-step RT-PCR – A" and " One-step RT-PCR with comparable performance to two-step RT-PCR – B", and table “One-step RT-PCR with comparable performance to two-step RT-PCR”).

One-step RT-PCR with comparable performance to two-step RT-PCR.

Amount of cDNA/RNA

Mean Ct value

(two-step RT-PCR)

Mean Ct value

(one-step RT-PCR)

100 ng 24.66 24.66
10 ng 28.15 27.84
1 ng 31.42 31.49
0.1 ng 34.80 34.56
0.01 ng 37.78 37.66

Sensitive detection of as few as 10 copies of target sequence (see figure " Detection of down to 10 copies of target RNA in duplex PCR") can be achieved in multiplex qRT-PCR with QuantiTect Multiplex RT-PCR Kits. Precise relative quantification of the expression of a gene is achieved by quantifying the expression of both the target gene and an endogenous control gene in the same well or tube. With QuantiTect Multiplex RT-PCR Kits, the CT values for the targets in a multiplex reaction are equivalent to those obtained in control experiments where the targets are amplified in separate reactions (see figure " Comparable amplification in triplex PCR and singleplex PCRs"). This demonstrates that the different targets in the same multiplex reaction are efficiently and sensitively amplified without affecting each other.

See figures

Principle

QuantiTect RT-PCR Kits contain an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification of RNA targets using SYBR Green I or sequence-specific probes. The fluorescent dye SYBR Green I in the QuantiTect SYBR Green RT-PCR master mix enables the analysis of many different targets without having to synthesize target-specific labeled probes (see table “Components of 2x QuantiTect SYBR Green RT-PCR Kit”).

Components of 2x QuantiTect SYBR Green RT-PCR Kit

Component Features Benefits
HotStarTaq DNA Polymerase 15 min activation at 95ºC Set-up of qPCR reactions at room temperature
QuantiTect SYBR Green RT-PCR Buffer Balanced combination of NH4+ and K+ ions Specific primer annealing ensures reliable PCR results
dNTP mix Includes dUTP, which partially replaces dTTP and enables optional UNG treatment of reactions Eliminates contamination from carryover of PCR products by optional UNG treatment
SYBR Green I dye Yields a strong fluorescent signal upon binding double-stranded DNA Highly sensitive quantification
ROX dye For normalization of fluorescent signals on Applied Biosystems and, optionally, Agilent instruments Precise quantification on cyclers that require ROX dye. Does not interfere with reactions on other real-time cyclers
Omniscript and Sensiscript Reverse Transcriptases Special blend of enzymes with high affinity for RNA RNA can be transcribed, even through complex secondary structures

QuantiTect Probe RT-PCR Kits contain an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification of RNA targets using sequence-specific probes (see table “Components of 2x QuantiTect Probe RT-PCR Kit”). The kits are designed for use with all types of sequence-specific probes, including hydrolysis probes (e.g., TaqMan® and other dual-labeled probes), FRET probes and Molecular Beacons.

Components of 2x QuantiTect SYBR Green RT-PCR Kit

Component Features Benefits
HotStarTaq DNA Polymerase 15 min activation at 95ºC Set-up of qPCR reactions at room temperature
QuantiTect Probe RT-PCR Buffer Balanced combination of NH4+ and K+ ions Specific primer annealing ensures reliable PCR results
dNTP mix Includes dUTP, which partially replaces dTTP and enables optional UNG treatment of reactions Eliminates contamination from carryover of PCR products by optional UNG treatment
SYBR Green I dye Yields a strong fluorescent signal upon binding double-stranded DNA Highly sensitive quantification
ROX dye For normalization of fluorescent signals on Applied Biosystems and, optionally, Agilent instruments Precise quantification on cyclers that require ROX dye. Does not interfere with reactions on other real-time cyclers
Omniscript and Sensiscript Reverse Transcriptases Special blend of enzymes with high affinity for RNA RNA can be transcribed, even through complex secondary structures

Precise relative quantification of the expression of a gene is achieved by quantifying the expression of both the target gene and an endogenous control gene in the same well or tube. The optimized master mix in the QuantiTect Multiplex RT-PCR Kit ensures that PCR products in a multiplex reaction are amplified with the same efficiency and sensitivity as PCR products in a corresponding single-amplification reactions (see table “Components of 2x QuantiTect Multiplex RT-PCR Kit”). As few as 10 copies of a target gene can be detected with the kit.

Components of 2x QuantiTect Multiplex RT-PCR Kit

Component Features Benefits
HotStarTaq DNA Polymerase 15 min activation at 95ºC Set-up of qPCR reactions at room temperature
QuantiTect Multiplex RT-PCR Buffer 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
dNTP mix Includes dUTP, which partially replaces dTTP and enables optional UNG treatment of reactions Eliminates contamination from carryover of PCR products by optional UNG treatment
ROX dye* For normalization of fluorescent signals on Applied Biosystems and, optionally, Agilent instruments Precise quantification on cyclers that require ROX dye. Does not interfere with reactions on other real-time cyclers
Omniscript and Sensiscript Reverse Transcriptases Special blend of enzymes with high affinity for RNA RNA can be transcribed, even through complex secondary structures

The balanced combination of K+ and NH4+ ions in the PCR buffer of QuantiTect RT-PCR Kits – plus unique synthetic Factor MP in the QuantiTect Multiplex RT-PCR buffer – promotes specific primer annealing, enabling high RT-PCR specificity and sensitivity (see figure " Specific primer annealing"). In addition, an optimized mix of reverse transcriptases enables cDNA synthesis from a wide range of RNA template amounts, while HotStarTaq DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.


QuantiTect RT-PCR master mixes also contain dUTP, enabling pretreatment with uracil-N-glycosylase (UNG) prior to starting PCR, which ensures that any contaminating PCR products do not affect subsequent PCR reactions.

See figures

Procedure

QuantiTect RT-PCR Kits overcome the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers and template to the ready-to-use QuantiTect SYBR Green RT-PCR master mix – or primers, probe and template to the ready-to-use QuantiTect Probe RT-PCR master mix – and start the reaction (see flowchart " One-step RT-PCR using SYBR Green" and “ One-step RT-PCR using sequence-specific probes"). Follow the protocol in the handbook to get fast and reliable results on any real-time cycler. If required, reactions can be pretreated with uracil-N-glycosylase (UNG) to eliminate carryover of PCR products from previous reactions.

Highly specific results in gene expression analysis are guaranteed when QuantiTect SYBR Green RT-PCR Kits are used in combination with QuantiTect Primer Assays. These are genomewide, bioinformatically validated primer sets for detecting transcripts from human, mouse, rat and many other species. QuantiTect Primer Assays can be easily ordered online at GeneGlobe.

The handbook for QuantiTect Multiplex RT-PCR Kits contains a single protocol that can be used with all available real-time cyclers and also lists recommended dyes. Kits are available with or without ROX passive reference dye in the master mix, enabling use on virtually any real-time cycler (see table “Choosing the right QuantiTect Multiplex RT-PCR Kit”). Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.

Choosing the right QuantiTect Multiplex RT-PCR Kit

ROX dye Kit Compatible cyclers
Supplied in master mix QuantiTect Multiplex RT-PCR Kit Cyclers from Applied Biosystems
Absent from master mix QuantiTect Multiplex RT-PCR NR Kit Rotor-Gene cyclers, and cyclers from Bio-Rad, Cepheid, Eppendorf, Roche, Agilent, and other suppliers
See figures

Applications

QuantiTect RT-PCR Kits can be used for 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, Rotor-Gene Multiplex RT-PCR Kit or Rotor-Gene SYBR Green RT-PCR Kit, which have been specially developed for fast cycling on these instruments.

Comparison of QuantiTect RT-PCR Kits

Features QuantiTect SYBR Green RT-PCR Kit QuantiTect Probe RT-PCR Kit QuantiTect Multiplex RT-PCR Kits
Applications Real-time quantification of RNA targets Real-time quantification of RNA targets Real-time quantification of RNA targets in a multiplex format
Reaction type One-step RT-PCR One-step RT-PCR Multiplex one-step RT-PCR
Real-time or endpoint Real time Real time Real time
Sample/target type RNA RNA RNA
Single or multiplex Single Single Single
SYBR Green I or sequence-specific probes SYBR Green I Sequence-specific probes Sequence-specific probes
Thermal cycler All real-time cyclers (e.g., LightCycler, Rotor-Gene, ABI) All real-time cyclers (e.g., LightCycler, Rotor-Gene, ABI) Real-time cyclers dedicated for multiplex PCR (e.g., most Applied Biosystems real-time cyclers, Roche LightCycler 480, and Bio-Rad iCycler iQ)
With or without ROX With ROX With ROX With ROX or without

Supporting data and figures

Resources

Brochures & Guides (1)
Kit Handbooks (4)
QuantiTect Multiplex PCR Kit - with master mix containing ROX passive reference dye QuantiTect Multiplex PCR NoROX Kit - with master mix free of ROX passive reference dye For quantitative, multiplex, real-time PCR and two-step RT-PCR using sequence-specific probes  
For quantitative, real-time one-step RT-PCR using SYBR Green I
For quantitative, real-time one-step RT-PCR using sequence-specific probes
For genomewide, ready-to-use real-time RT-PCR assays using SYBR Green detection

FAQ

Why does my PCR product show up later when comparing the QuantiTect SYBR Green PCR Kits with Roche kits using the same annealing temperature?
QuantiTect SYBR Green PCR Kits contain buffers with different salt concentrations compared to Roche Kits, resulting in different template melting temperatures. We recommend to decrease the annealing temperature by at least by 5°C when using a QuaniTect SYBR Green PCR Kit.
FAQ ID -1083
Does SYBR Green dye present in QuantiTect kits interfere with ethidium bromide staining?
SYBR Green dye does not interfere with ethidium bromide staining, and realtime SYBR Green PCR products can be visualized on an agarose gel.
FAQ ID -312
Do you have any information or guidelines regarding the choice of reference genes for real-time PCR?

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 [2002]: 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 [2009]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.

FAQ ID -2371
Do you recommend 1-step or 2-step real-time RT-PCR for gene expression analysis?

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:

Two-step RT-PCR One-step RT-PCR
Multiple PCRs from one RT reaction Easy handling
Flexibility with RT primer choice Fast procedure
Enables long-term storage of cDNA High reproducibility
  Low contamination risk

 

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'.

FAQ ID -1056
Can Uracil-N-Glycosylase (UNG) be used with a one-step RT-PCR kit, such as the QuantiTect Probe RT-PCR Kit?

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.

 

 

FAQ ID -2135
Can I do 4-plex real-time PCR, on the ABI PRISM 7000, 7700, or 7900 using the QuantiTect Multiplex PCR Kit?
No. Due to hardware limitations, the maximum capacity of these real-time cyclers is triplex PCR.
FAQ ID -716
Why is the reaction volume for QuantiFast PCR Kits lower than that for QuantiTect PCR Kits?

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

 

FAQ ID -1447
Why are the QuantiTect and QuantiFast Multiplex PCR Kits limited to triplex real-time PCR on some cyclers?

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.

FAQ ID -715
Why do replicates in real-time PCR have different plateau heights?

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".

 

FAQ ID -539
How should fluorescent labeled probes be stored?

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.

FAQ ID -784
Why is an ice-incubation step included during reaction set-up when following the QuantiTect RT-PCR but not the QuantiTect PCR protocol.
The ice step is recommended to prevent non-specific reverse transcription during reaction set-up. Mispriming by Taq is not a concern in these cases because both QuantiTect RT-PCR and PCR kits contain HotStarTaq, a DNA polymerase that only becomes active following a 15 minute, 95 degree incubation step.
FAQ ID -283
Do the master mixes in Rotor-Gene Kits contain dUTP to allow UNG pretreatment?

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.

 

 

FAQ ID -2117
If template abundance differs significantly between housekeeping and target gene, are both amplified equally efficient with the QuantiTect Multiplex Kits?
Yes. The pre-optimized master mix ensures that all gene targets in a multiplex reaction are amplified with the same efficiency and sensitivity as in corresponding "single-plex" reactions, independent of starting copy numbers.
FAQ ID -718
How do I create a workspace that is free of DNA contamination, prior to carrying out a qPCR experiment?

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.  
FAQ ID -2654
How long should the amplicon be if I am using QuantiTect SYBR Green detection?
For accurate quantification we recommend an amplicon length of 100-150 bp to ensure optimal amplification efficiency.
FAQ ID -553
What reaction volume should I use with QuantiTect Kits?
QuantiTect Kits for real-time PCR and RT-PCR have been optimized for a final reaction volume of 50 µl for use with single tubes and 96-well plates, or 20 µl for use with 384-well plates and LightCycler capillaries. We strongly recommend using the primer and probe concentrations, reaction volumes, and amount of starting template given in the QuantiTect handbooks. However, volumes may be reduced to 20 µl and 10 µl, respectively.
FAQ ID -551
Do you have information on the use of recombinant DNA and RNA as absolute standards for realtime RT-PCR?

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.

FAQ ID -729
What is a QuantiTect Primer Assay?

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.

FAQ ID -1141
What is the concentration of the primers in a reaction using the QuantiTect Primer Assays?

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.

FAQ ID -850
Can the Reverse Transcriptases of the QuantiTect Reverse Transcription Kit and the QuantiTect Probe RT-PCR Kit be used interchangeably?

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.

 

FAQ ID -1066
My QuantiTect Master Mix did not freeze at degrees. Is it still ok to use?
Yes. The QuantiTect Master Mix may not necessarily freeze at -20 degrees. The performance is not affected as long as the correct storage temperature is maintained.
-20
What is a quenched FRET assay?
Quenched FRET assays are similar to FRET assays except that the decrease in energy of the donor fluorophore is measured instead of the increase in energy of the acceptor fluorophore.
FAQ ID -542
What annealing temperature should be used with the QuantiTect Primer Assays?

The annealing temperature for QuantiTect Primer Assays should be 55oC 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 60oC, as described in the QuantiTect Primer Assay Handbook.

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

 

 

FAQ ID -849
Why is the RT step with the QuantiFast RT Kits much shorter compared to QuantiTect RT Kits?

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.

 

FAQ ID -1451
Why is the storage time for QuantiFast PCR Kits shorter than that for QuantiTect PCR Kits?

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.

 

FAQ ID -1446
Have QuantiTect Primer Assays been tested with QuantiFast SYBR Green PCR Kits on the Mastercycler ep realplex?

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.

 

FAQ ID -1714
Can Uracil-N-Glycosylase (UNG) be purchased separately, independent of QuantiTect PCR Kits?
No, UNG is only available in combination with a QuantiTect PCR Kit.
FAQ ID -2134
How important is the RNA purification process, for obtaining reliable qRT-PCR results?

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.

FAQ ID -2655
What is the threshold cycle or Ct value?
The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.
FAQ ID -2682
Why are my realtime PCR amplification plots hook-shaped?
During the late phase of PCR, as a lot of PCR product has been generated, there is strong competition between hybridization of the probe to the target strand and re-association of the two complementary product strands. For some primer–probe combinations, re-association occurs more quickly than probe hybridization towards the end of the PCR. Therefore, the yield of PCR product seems to decrease after reaching a peak. For accurate quantification, fluorescence data is measured during the log-linear phase of the reaction, i.e., during the initial increase of fluorescence, before this phenomenon occurs.
FAQ ID -587
QuantiTect Primer Assays are bioinformatically validated, genomewide primer sets. What does “bioinformatically validated” mean?

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.

 

FAQ ID -1982
Do I need to determine limiting primer concentrations with the QuantiTect Multiplex PCR Kits?
No, that is not necessary. Simply use the primer concentrations specified in the protocols in the supplied QuantiTect Multiplex PCR handbooks.
FAQ ID -714
Can I increase the annealing temperature recommended for QuantiTect Primer Assays used with the QuantiTect SYBR Green PCR Kits?

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.

 

FAQ ID -1146
Do you have a protocol for performing quantitative multiplex PCR on the LightCycler 2.0 system?
Do you have a protocol for adding new reporter dyes to the ABI PRISM 7900 Sequence Detection System?
Why does my realtime PCR assay quality decrease over time?
Make sure that template, primers, probes, and amplification reagents are stored correctly and avoid multiple freeze–thaw cycles for oligonucleotides and template. Check the performance of your real-time instrument as some instruments require the halogen lamp to be frequently replaced. Lasers must also be replaced occasionally.
FAQ ID -589
Which real-time PCR kits are recommended downstream of the QuantiTect Whole Transcriptome Kit?

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

 

FAQ ID -1592
How much time will be saved when switching from standard cycling to fast cycling with QuantiFast Kits?

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%.

 

 

FAQ ID -1438
How much template can I use in the reaction and what is the maximum volume of template that can be used in the QuantiTect Kits?

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.

FAQ ID -1086
How can one perform multiplex real-time PCR analysis using the LightCycler 2.0?
The LightCycler 2.0 requires a color compensation file to separate the fluorescent signals and eliminate crosstalk between the individual detection channels. Please contact QIAGEN Technical Services for a specialized protocol.
FAQ ID -780
Do QuantiTect Primer Assays contain SYBR Green dye?

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

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

FAQ ID -1143
Where can I find info on compatible reporter dyes for use in real-time multiplex PCR using the QuantiTect Multiplex PCR Kits?

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

 

 

 

FAQ ID -719
Do the QuantiTect SYBR Green Kits contain a stabilization reagent that inhibits the sensitivity of real-time PCR?
No. In fact, QuantiTect SYBR Green PCR Kits contain a stabilization reagent that increases the sensitivity of real-time PCR. The QuantiTect SYBR Green Kits also contain a carefully controlled concentration of SYBR Green that is sufficient to detect PCR fragments while having no inhibitory effect on the enzymatic reaction.
FAQ ID -328
Why should DNA or cDNA targets be less than 250 bp long for real-time PCR?

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.

FAQ ID-751
What should I use as a standard for absolute quantification 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.

FAQ ID -1085
What are the main differences between Rotor-Gene and QuantiTect or QuantiFast PCR Kits?

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.

 

FAQ ID -2119
Can I make a master mix with QuantiProbes, primers, and all other reaction components and store it for later use?

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.

FAQ ID -584
How do QuantiFast PCR Kits compare to QuantiTect PCR Kits for quantitative real-time PCR?

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.

 

 

FAQ ID -1441
Is it possible to perform a UNG treatment when using QuantiTect kits?

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.

FAQ ID -564
Can I use the QuantiTect Multiplex PCR Kits on the Roche LightCycler systems with TaqMan® probes or QuantiTect Assays?
  • 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.

FAQ ID -717
Do I need to calibrate my real-time cycler if I want to use Yakima Yellow?
The emission maximum of Yakima Yellow (552 nm) is almost identical to that of the fluorescent dye VIC. Therefore, the channel and filter settings for VIC can also be used for Yakima Yellow.
FAQ ID -540
Why do melting temperatures differ between PCR fragments amplified with QIAGEN's QuantiTect SYBR Green PCR Kits and Roche Kits?
The melting temperature of a PCR product depends on the salt concentration of the buffers used for amplification. Buffer compositions differ between QuantiTect SYBR Green PCR Kits and Roche kits, resulting in different PCR product melting temperatures.
FAQ ID -1084