QuantiTect RT-PCR Kits

• 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 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.

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.

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.

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

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 Probe RT-PCR Kit

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

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.

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

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.