RT-PCR Dual Probe Synthesis

For one-step qRT-PCR using sequence-specific probes for gene expression analysis

• 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
• No need to optimize reaction and cycling conditions

QuantiTect Probe RT-PCR Kits enable sensitive quantification of RNA targets by real-time one-step 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 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 mix can be stored at 2–8°C.
One-step RT-PCR with comparable performance to two-step…Tenfold serial dilutions of cDNA (100 ng to 1 pg) prepared from leukocytes were analyzed in duplicate on the Mx3005 using primers and a FAM labeled probe specific for IL1R2 (interleukin 1 receptor, type II). Two-step RT-PCR was performed using the QuantiTect Probe PCR Kit (PCR efficiency: 101%).

Performance

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. It contains HotStarTaq DNA Polymerase, which provides the most stringent hot start compared with other polymerases (see figure “Highly specific amplification”).

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 C_T 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).
Principle

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. 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. QuantiTect Probe RT-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, 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 Probe RT-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.

Procedure
The QuantiTect Probe RT-PCR Kit overcomes the need for optimization of reaction conditions, which can be tedious and time-consuming. Simply add primers, probe, and RNA template to the ready-to-use RT-PCR master mix, and start the reaction (see flowchart “One-step RT-PCR”).