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

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 .

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.

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.

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 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 . This demonstrates that the different targets in the same multiplex reaction are efficiently and sensitively amplified without affecting each other.

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 。

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 . 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 . Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.

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.