For qPCR and two-step qRT-PCR using SYBR Green I and for qPCR and two-step qRT-PCR and also multiplex qPCR and two-step multiplex qRT-PCR using sequence-specific probes

Features

Highly sensitive detection of low-copy targets

qPCR using sequence-specific probes or SYBR Green

Accurate quantification over several logs of template

No need to optimize reaction and cycling conditions

Detection of up to 5 targets in the same tube

Product Details

QuantiTect PCR Kits (qPCR kits) enable sensitive quantification of gDNA and cDNA targets by real-time PCR and two-step RT-PCR using sequence-specific probes or SYBR Green I detection. The real-time PCR kits also allow 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 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 mixes in QuantiTect PCR Kits can be stored at 2–8°C.

Two kit formats are available for multiplex PCR using sequence-specific probes: 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.

Performance

HotStarTaq DNA Polymerase included in QuantiTect quantitative PCR kits increases the specificity of the PCR reaction by providing the most stringent hot start compared with other polymerases.

QuantiTect SYBR Green PCR Kits allow specific quantification over a wide linear range  When used in combination with the QuantiTect Reverse Transcription Kit and QuantiTect Primer Assays, QuantiTect SYBR Green PCR Kits deliver sensitive and reliable results.

QuantiTect Probe PCR Kits deliver sensitive and reliable results in combination with the QuantiTect Reverse Transcription Kit. 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.

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 .

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 .

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

Principle

QuantiTect SYBR Green PCR Kits contain an optimized, ready-to-use master mix for highly specific and sensitive real-time quantification cDNA targets using SYBR Green I . The fluorescent dye SYBR Green I in the master mix enables the analysis of many different targets without having to synthesize target-specific labeled probes. A balanced combination of K+ and NH4+ ions in the PCR buffer promotes specific primer annealing and enables high PCR specificity and sensitivity. In addition, HotStarTaq DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.

Procedure

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.

QuantiTect SYBR Green and Probe PCR Kits overcome the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers and DNA template to the ready-to-use PCR master mix, and start the reaction. Follow the protocol in the handbook to get fast and reliable results on any real-time cycler.

Highly specific results in gene expression analysis are guaranteed when QuantiTect SYBR Green 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.

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.

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

Applications

QuantiTect 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 SYBR Green PCR Kit, Rotor-Gene Probe PCR Kit or Rotor-Gene Multiplex PCR Kit, which have been specially developed for fast cycling on these instruments.

Supporting data and figures

Specific quantification over a wide linear range.

Dilutions of human leukocyte cDNA were analyzed using the QuantiTect Primer Assay for human BAX and the indicated kits on the Rotor-Gene 3000. Highly specific quantification with the QuantiTect Kit and Assay is demonstrated by the single peak in melting curve analysis and the flat curve for the NTC reaction. NTC: No template control. Norm. Fluoro.: Normalized fluorescence.