For real-time PCR and two-step RT-PCR using sequence-specific probes

  • Highly sensitive detection of low-copy targets
  • Accurate quantification over several logs of template
  • Use of any sequence-specific probe on any real-time cycler
  • Available with or without uracil-N-glycosylase (UNG)
  • No need to optimize reaction and cycling conditions

QuantiTect Probe PCR Kits enable sensitive quantification of gDNA and cDNA targets by real-time PCR and two-step RT-PCR using sequence-specific probes. The combination of a hot start and a unique PCR buffer system in the ready-to-use master mix ensures highly sensitive qPCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. For convenience, the master mix can be stored at 2–8°C.

Quantity Product Cat. no. Price
 
 
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  varies
Quantity Product Cat. no. Price Sum
 
QuantiTect Probe PCR Kit (40)
Trial kit for 40 x 50 µl reactions: 1 ml 2x QuantiTect Probe PCR Master Mix, 2 ml RNase-Free Water
204341
$117.00
$0.00
 
QuantiTect Probe PCR Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Probe PCR Master Mix, 2 x 2 ml RNase-Free Water
204343
$443.00
$0.00
 
QuantiTect Probe PCR Kit (1000)
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect Probe PCR Master Mix, 20 ml RNase-Free Water
204345
$1,767.00
$0.00
 
QuantiTect Probe PCR +UNG Kit (200)
For 200 x 50 µl reactions: 3 x 1.7 ml 2x QuantiTect Probe PCR Master Mix, 100 ul UNG Solution, 2 x 2 ml RNase-Free Water
204363
$510.00
$0.00

QuantiTect Probe 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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Effective UNG digestion.|Two-step RT-PCR.|Highly specific amplification.|Wide dynamic range in two-step PCR.|Wide dynamic range in real-time PCR.|High sensitivity and efficiency, and wide dynamic range.|Specific primer annealing.|
106 copies of two dUMP-containing PCR amplicons were treated with or without UNG and then amplified by real-time PCR. UNG was from various suppliers, and real-time PCR was performed using the master mix from the QuantiTect Probe PCR +UNG Kit. ΔCT on the Y-axis indicates CT values for non-UNG-treated samples subtracted from CT values for UNG-treated samples. The UNG supplied with the QuantiTect Kit provided the greatest ΔCT (9-12 cycles). This indicates that QIAGEN UNG digests carryover PCR products more effectively than UNG from other suppliers.|The QuantiTect Probe PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers, probe, and cDNA template to the ready-to-use PCR master mix, and start the reaction on any real-time cycler (see also the table "Components of 2x QuantiTect Probe PCR Master Mix").|In comparison with other DNA polymerases, only HotStarTaq DNA Polymerase in combination with a unique buffer specifically amplified a 497 bp fragment (from 50 copies of an HIV-pol-gene construct in a background of 1 µg human genomic DNA). M: Markers.|Duplicate reactions were performed on the Mx3005P using tenfold dilutions of human keukocyte cDNA (10 ng to 0.01 ng) and a TaqMan assay for IL1R2 (a cytokine). The QuantiTect Probe PCR Kit provided accurate gene expression analysis from low to high template amounts with a PCR efficiency of 101%.|Probe-based real-time PCR with UNG pretreatment was carried out using the QuantiTect Probe PCR +UNG Kit. Reactions were run in duplicate on the ABI PRISM 7900 using 10-fold dilutions of human leukocyte cDNA (100 ng to 10 pg) and a TaqMan assay for IL8 (a cytokine). The amplification plots were evenly spaced, leading to a high PCR efficiency of 94%.|Tenfold serial dilutions of leukocyte cDNA (100 ng to 1 pg) were analyzed in duplicate on the Mx3005 using the QuantiTect Probe PCR Kit with primers and a FAM-labeled probe specific for IL8 (interleukin 8). A PCR efficiency of 97% over 6 logs of template dilution was achieved, and as little as 1 pg of IL8 transcript was sensitively detected.| A balanced combination of KCl and (NH4)2SO4 promotes specific annealing of primers and probes to the PCR template. K+ binds to phosphate groups on double-stranded DNA, stabilizing annealing of primers and probes. NH4+ destabilizes weak hydrogen bonds between mismatched bases. |
Performance

The QuantiTect Probe PCR Kit delivers sensitive and reliable results in combination with the QuantiTect Reverse Transcription Kit (see figure "High sensitivity and efficiency, and wide dynamic range"). The unique PCR buffer composition enables QuantiTect Probe PCR Kits to provide sensitive quantification of low-copy DNA targets, as well as accurate quantification over a wide linear range (see figures "High sensitivity and efficiency, and wide dynamic range" and "Wide dynamic range in two-step RT-PCR"). HotStarTaq DNA Polymerase further increases the specificity of the PCR reaction by providing the most stringent hot start compared with other polymerases (see figure "Highly specific amplification").

A major source of PCR contamination is the carryover of PCR products from previous reactions. Pretreatment with UNG prior to starting PCR ensures that any contaminating PCR products do not affect subsequent PCR. The combination of the specially optimized UNG solution and the proven PCR master mix in the QuantiTect Probe PCR +UNG Kit ensures effective elimination of carried-over PCR products together with reliable quantification of target sequences (see figures "Effective UNG digestion" and "Wide dynamic range in real-time PCR").

Principle

QuantiTect Probe PCR Kits contain an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification of gDNA and cDNA targets using sequence-specific probes. The kits are designed for use with all types of sequence-specific probes, including hydrolysis probe detection (e.g., TaqMan and other dual-labeled probes), FRET probes, and Molecular Beacons. QuantiTect Probe PCR Kits contain a unique PCR buffer that contains a balanced combination of K+ and NH4+ ions, which promote specific primer annealing, enabling high PCR specificity and sensitivity (see figure "Specific primer annealing"). In addition, HotStarTaq DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

QuantiTect Probe 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 Probe PCR Kit
ComponentFeatures Benefits
HotStarTaq DNA Polymerase 15 min activation at 95ºC Set-up of qPCR reactions at room temperature
QuantiTect Probe 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
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
Procedure

QuantiTect Probe PCR Kits overcome the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers, probe, and DNA template to the ready-to-use PCR master mix, and start the reaction (see flowchart "Two-step RT-PCR"). 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. 

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 Probe PCR Kits can be used for probe-based gene expression analysis of cDNA targets or quantitative gDNA analysis 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 PCR Kit, which has been specially developed for fast cycling on these instruments.

Feature
Specifications
Applications Real-time quantification of DNA, cDNA, or RNA targets
Reaction type PCR and two-step RT-PCR
Real-time or endpoint Real-time
Sample/target type DNA, cDNA, RNA
Single or multiplex Single
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

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Kit Handbooks
4
配列特異的プローブを用いたリアルタイム定量PCRおよび2ステップRT-PCR用
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PCR産物のキャリーオーバーを除去するためにQuantiTect® PCR Kitsと併用
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For quantitative, real-time PCR and two-step RT-PCR using sequence-specific probes
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For use with QuantiTect PCR Kits to eliminate carryover of PCR products
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