For multiplex, real-time PCR and RT-PCR using sequence-specific probes

  • Multiplex analysis with no need for optimization
  • Sensitive detection of as few as 10 copies of each target
  • Reliable quantification of target and reference genes
  • Detection of up to 5 targets in the same tube

QuantiTect Multiplex PCR Kits enable reliable quantification of up to 5 gDNA or cDNA targets in a single tube by multiplex, real-time PCR or two-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 and reliable multiplex qPCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. Two kit formats are available: the QuantiTect Multiplex PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiTect Multiplex PCR NoROX Kit for all other cyclers. For convenience, the master mix can be stored at 2–8°C.

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Quantity Product Cat. no. Price Sum
 
QuantiTect Multiplex PCR Kit (40)
Trial kit for 40 x 50 µl reactions: 1 ml 2x QuantiTect Multiplex PCR Master Mix (with ROX dye), 2 ml RNase-Free Water
204541
$159.00
$0.00
 
QuantiTect Multiplex PCR Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Multiplex PCR Master Mix (with ROX dye), 2 x 2 ml RNase-Free Water
204543
$666.00
$0.00
 
QuantiTect Multiplex PCR Kit (1000)
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect Multiplex PCR Master Mix (with ROX dye), 20 ml RNase-Free Water
204545
$2,668.00
$0.00
 
QuantiTect Multiplex PCR NoROX Kit (40)
Trial kit for 40 x 50 µl reactions: 1 ml 2x QuantiTect Multiplex PCR NoROX Master Mix (without ROX dye), 2 ml RNase-Free Water
204741
$159.00
$0.00
 
QuantiTect Multiplex PCR NoROX Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Multiplex PCR NoROX Master Mix (without ROX dye), 2 x 2 ml RNase-Free Water
204743
$666.00
$0.00
 
QuantiTect Multiplex PCR NoROX Kit (1000)
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect Multiplex PCR NoROX Master Mix (without ROX dye), 20 ml RNase-Free Water
204745
$2,668.00
$0.00

QuantiTect Multiplex PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.


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Equivalent CT values in triplex and singleplex PCR.|Comparable results between 4-plex PCR and corresponding singleplex PCRs.|Successful quantitative triplex qPCR of low and high abundance targets.|Detection of 10 copies of target gene with excess reference gene.|QIAGEN multiplex kits.|Unique PCR buffer.|
Dilutions of cDNA template (100 ng, 11.11 ng, 1.23 ng, and 0.14 ng) were analyzed by triplex PCR (colored curves) and by singleplex PCR (gray curves) on the iCycler iQ. TaqMan probes labeled with FAM, HEX, or Cyanine 670 dye were used. (Data kindly provided by the University of Minnesota, Minneapolis, MN, USA.)|GAPDH, 18S, HSP, and H28S sequences were amplified either together in 4-plex PCR or in singleplex PCRs on the Rotor-Gene 3000 system using the QuantiTect Multiplex PCR NoROX Kit. The templates were 100, 10, 1, and 0.1 ng of human leukocyte cDNA. Reactions were performed in triplicate. TaqMan probes labeled with FAM, HEX, Texas Red, or Cyanine 670 dye were used. 4-plex PCR and singleplex PCR data are displayed on the amplification plots, with the curves for the singleplex PCRs colored in gray. The plots show reliable multiplex quantification over a wide range of template amounts as well as good reproducibility within each set of triplicates. GAPDH: glyceraldehyde-3-phosphate dehydrogenase; 18S: 18S rRNA; HSP: a heat shock protein; H28S: 28S rRNA.|Three sequences (CSBG, GAPDH, and HSP) were amplified either together in triplex PCR or in singleplex PCRs on the LightCycler 2.0 system using the QuantiTect Multiplex PCR NoROX Kit. Three template mixes with varying amounts of human genomic DNA (for CSBG amplification), linearized plasmid (for GAPDH amplification), and human leukocyte cDNA (for HSP amplification) were prepared. Reactions were performed in duplicate. TaqMan probes labeled with FAM, HEX, or Texas Red dye were used. The amplification plot shows detection of CSBG in the triplex PCRs (Blue: 1400 copies; Green: 140 copies; Red: 14 copies).|[A] Duplex PCR was performed on the ABI PRISM 7700 using either the QuantiTect Multiplex PCR Kit or a kit from Supplier AII. Variable amounts of t(8;14) translocation sequence (50,000, 5000, 500, 50, or 10 copies) were coamplified with a constant amount of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) sequence (107 copies). The t(8;14) translocation sequence was amplified from Ramos cell line genomic DNA using a FAM labeled TaqMan probe. The GAPDH sequence was amplified from a plasmid containing GAPDH cDNA sequence using a HEX labeled TaqMan probe. In contrast to the kit from Supplier AII, the QuantiTect Multiplex PCR Kit reliably quantified high to low amounts of t(8;14) translocation sequence (down to 10 copies, indicated with an arrow) in the presence of a high amount (107 copies) of GAPDH sequence. [B] The procedure described in [A] was repeated, with the exception that the target sequences were amplified separately in singleplex PCRs.|QuantiTect Multiplex PCR Kits provide a simple procedure for quantitative, multiplex, real-time PCR. In contrast to current methods, the kits eliminate the need for optimization of the concentrations of primers, Mg2+, and Taq DNA polymerase, even for difficult assays (e.g., assays in which the copy number of the target gene is much smaller than that for the reference gene).|[A] NH4+ ions prevent nonspecific primers from annealing to the template. [B] Synthetic Factor MP, an innovative PCR additive, increases the local concentration of primers at the template. Together with K+ and other cations, synthetic Factor MP stabilizes specifically bound primers, allowing efficient primer extension by HotStarTaq DNA Polymerase.|
Performance

The QuantiTect Multiplex PCR Master Mix ensures that the PCR products in a multiplex reaction are amplified with the same efficiency and sensitivity as the PCR products in the corresponding single-amplification reactions (see table “Comparable threshold cycle (CΤ) values with triplex and singleplex PCRs”, and figures “Comparable results with 4-plex and singleplex PCRs” and "Equivalent CT values in triplex PCR and singleplex PCR").

Comparable threshold cycle (CT) values with triplex and singleplex PCRs
Detection of t(8;14) translocation sequence (20 copies) Detection of GAPDH cDNA sequence (10copies) Detection of NFKB cDNA sequence (see first column for copy number)
Triplex PCR with 105 copies of NFKB
34.31 20.37 21.92
Corresponding singleplex PCRs    34.07 20.54 21.83
Triplex PCR with 104 copies of NFKB 34.61 20.62 25.03
Corresponding singleplex PCRs 34.00 20.46 25.19
Triplex PCR with 103 copies of NFKB 35.17 19.94 28.38
Corresponding singleplex PCRs 34.43 20.50 28.65
Three sequences were amplified either together in triplex PCR or in singleplex PCRs on the ABI PRISM 7900 using the QuantiTect Multiplex PCR Kit. The templates were 140 pg of genomic DNA from the Ramos cell line carrying a t(8;14) translocation (about 20 copies of the target sequence); 106 copies of a linearized plasmid containing human GAPDH cDNA sequence (glyceraldehyde-3-phosphate dehydrogenase); and 105, 104, or 103 copies of a plasmid containing human NFKB cDNA sequence (nuclear factor of kappa light polypeptide gene enhancer in B-cells). This simulates quantification of low, high, and variable amounts of target genes in a single assay. TaqMan probes labeled with FAM, HEX, or Bodipy TMR reporter dye plus BHQ quencher were used.

As few as 10 copies of a target gene can be detected with the kits, even if the copy number of the reference gene in the same reaction is up to 106-fold greater (see figures “Detection of 10 copies of target gene with excess reference gene", "Successful quantitative triplex qPCR of low and high abundance targets" and the corresponding table "Successful quantitative triplex qPCR of low and high abundance targets").

Successful quantitative triplex real-time PCR of low and high abundance targets
Detection of CSBG Detection of GAPDH Detection of HSP
Template mix 1 1400 copies 106 copies 2 x 104 copies
CT values for triplex PCR 27.73 18.69 23.59
CT values for singleplex PCRs 27.08 18.89 23.52
Template mix 2 140 copies 106 copies 2 x 103 copies
CT values for triplex PCR 31.11 19.00 27.05
CT values for singleplex PCRs 30.66 18.61 26.97
Template mix 3 14 copies 106 copies 2 x 102 copies
CT values for triplex PCR 34.74 18.98 30.71
CT values for singleplex PCRs 33.84 19.01 30.38
Three sequences (CSBG, GAPDH, and HSP) were amplified either together in triplex PCR or in singleplex PCRs on the LightCycler 2.0 system using the QuantiTect Multiplex PCR NoROX Kit. Three template mixes with varying amounts of human genomic DNA (for CSBG amplification), linearized plasmid (for GAPDH amplification), and human leukocyte cDNA (for HSP amplification) were prepared. Reactions were performed in duplicate. TaqMan probes labeled with FAM, HEX, or Texas Red dye were used. The table shows the approximate copy numbers of the targets and the mean threshold cycle (CΤ) values for the triplex and singleplex PCRs. The data demonstrate comparable CΤ values between the triplex and singleplex PCRs as well as detection of as little as 14 copies of CSBG sequence in the presence of a high amount (106 copies) of GAPDH sequence. CSBG: Candida solubilized β-glucan; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HSP: a heat shock protein.
Principle

QuantiTect Multiplex PCR Kits enable success in multiplex, two-step RT-PCR on the first attempt (see flowchart "QIAGEN multiplex kits"). The optimized master mix 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. As few as 10 copies of a target gene can be detected with the kit.

Amplifying reference and target genes in the same reaction instead of in separate reactions increases the reliability of gene quantification by minimizing handling errors. The QuantiTect Multiplex PCR Master Mix contains 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 PCR buffer"). In addition, HotStarTaq DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

The QuantiTect Multiplex PCR Master Mix also contains 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.

Components of 2x QuantiTect Multiplex PCR Kit
ComponentFeatures Benefits
HotStarTaq DNA Polymerase 15 min activation at 95ºC Set-up of qPCR reactions at room temperature
QuantiTect Multiplex 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* Normalizes 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
* ROX dye is either present in the master mix or not. See table "Choosing the right QuantiTect Multiplex PCR Kit".
Procedure

QuantiTect Multiplex PCR Kits contain ready-to-use master mixes that eliminate the need for optimization of reaction and cycling conditions. The handbook contains a single protocol that can be used with all available real-time cyclers and also lists recommended dyes. If required, reactions can be pretreated with uracil-N-glycosylase (UNG) (not supplied) to eliminate carryover of PCR products from previous reactions. 

Kits are available with or without ROX passive reference dye in the master mix, enabling use on virtually any real-time cycler (see table).  Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.

Choosing the right QuantiTect Multiplex PCR Kit
ROX dye Kit Compatible cyclers
Supplied in master mix QuantiTect Multiplex PCR Kit Cyclers from Applied Biosystems
Absent from master mix QuantiTect Multiplex PCR NoROX Kit Rotor-Gene cyclers, and cyclers from Bio-Rad, Cepheid, Eppendorf, Roche, Agilent, and other suppliers

For optimal results in real-time two-step RT-PCR, we recommend synthesizing cDNA using the QuantiTect Reverse Transcription Kit. The kit provides fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination.

Applications

QuantiTect Multiplex PCR Kits can be used for gene expression analysis of cDNA or quantification of gDNA 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 Multiplex PCR Kit, which has been specially developed for fast cycling on these instruments.

Feature
Specifications
Applications Quantification of cDNA or genomic DNA targets
Reaction type PCR product amplification
Real-time or endpoint Real-time
Sample/target type cDNA, DNA
Single or multiplex Multiplex
SYBR Green I or sequence-specific probes Sequence-specific probes
Thermal cycler Most real-time cyclers (e.g. LC, RG, ABI)
With or without ROX With ROX or without

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Kit Handbooks
2
QuantiTect Multiplex PCR Kit ̶ ROX passive reference dye を含むマスターミックス添付
QuantiTect Multiplex PCR NoROX Kit ̶ ROX passive reference dye を含まないマスターミックス添付
配列特異的プローブを用いたマルチプレックス・リアルタイム定量PCR および2 ステップのRT-PCR
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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  
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