QuantiTect PCR and RT-PCR System

QIAGEN QuantiTect PCR Kits (for PCR and two-step RT-PCR) and QuantiTect RT-PCR Kits (for one-step RT-PCR) provide accurate real-time quantification of DNA, cDNA, and RNA targets in an easy-to-handle format for any real-time thermal cycler.

Features and benefits

Products

Principle

The QuantiTect Kits provide reagents in an easy-to-use master-mix format. QuantiTect SYBR Green PCR Master Mix and QuantiTect SYBR Green RT-PCR Master Mix in QuantiTect SYBR Green Kits contain SYBR Green I, HotStarTaq DNA Polymerase, ROX, and a dNTP mix (including dUTP) in an optimized buffer. QuantiTect Probe PCR Master Mix and QuantiTect Probe RT-PCR Master Mix have been specifically adapted for quantitative PCR and RT-PCR analyses using sequence-specific probes.

In addition, QuantiTect RT-PCR Kits include QuantiTect RT Mix with an optimized blend of Omniscript and Sensiscript Reverse Transcriptases. Both enzymes exhibit a high affinity for RNA, facilitating transcription through secondary structures that may inhibit other reverse transcriptases.

QuantiTect Probe Kits are designed for use with all types of sequence-specific probes, including TaqMan or QIAGEN Operon dual-labeled probes, LightCycler hybridization (FRET) probes, and Molecular Beacons.

SYBR Green I in QuantiTect SYBR Green Kits yields a strong fluorescent signal on binding double-stranded DNA, providing rapid and accurate analysis of many different targets. Synthesis of sequence-specific probes is not required.

HotStarTaq DNA Polymerase in the master mix of all QuantiTect kits provides a hot start to the PCR for high specificity. The hot start prevents the formation of primer-dimers and nonspecific products. This leads to increased sensitivity, allowing detection of low-copy targets that could otherwise be obscured by high background levels, and avoiding problems with SYBR Green binding to nonspecific products (see figure "Highly Specific Real-Time PCR Using QuantiTect SYBR Green Kits").

Highly Specific Real-Time PCR Using QuantiTect SYBR Green Kits

 A  Agarose gel electrophoresis and melting curve analyses of reactions carried out using the LightCycler System. PCR products were amplified from 5 copies of a plasmid containing a fragment of the cystic fibrosis transmembrane regulator (CFTR) gene, using a PCR kit from Supplier R (R) or the QuantiTect SYBR Green PCR Kit from QIAGEN (Q). RT-PCR products were amplified from the low-density lipoprotein receptor (LDLR) transcript in 100 ng HeLa RNA, using an RT-PCR kit from Supplier R (R) or the QuantiTect SYBR Green RT-PCR Kit from QIAGEN (Q). M: markers.  B  Agarose gel electrophoresis and melting curve analyses of reactions carried out using the ABI PRISM 7700 and the GeneAmp 5700 Detection Systems. PCR products were amplified from 5 copies of a plasmid containing a fragment of the CFTR gene, using a PCR kit from Supplier AII (AII) or the QuantiTect SYBR Green PCR Kit from QIAGEN (Q). RT-PCR products were amplified from the low-density lipoprotein receptor (LDLR) transcript in 100 ng HeLa RNA, using an RT-PCR kit from Supplier AII (AII) or the QuantiTect SYBR Green RT-PCR Kit from QIAGEN (Q). M: markers.

The fluorescent dye ROX, included in the master mixes, serves as an internal reference for normalization of the fluorescent signal. This normalization is necessary for reactions on the ABI Sequence Detection Systems but does not interfere with quantification using other real-time PCR cyclers.

dNTPs in the master mixes include dUTP, which partially replaces dTTP. This allows an optional uracil-N-glycosylase (UNG) treatment of the reaction, which can be used if contamination with carried-over PCR products is suspected.

The QuantiTect SYBR Green and QuantiTect Probe PCR and RT-PCR buffers are specifically designed for quantitative PCR or RT-PCR using SYBR Green or sequence-specific probes, respectively. The buffers contain the balanced combination of cations included in all QIAGEN PCR and RT-PCR kits to promote specific annealing over a wide temperature range. In addition, the magnesium concentration provided in the kits is finely adjusted for optimal results, so the user does not need to experiment with different concentrations for each new target.

Procedure

QuantiTect Kits contain a ready-to-use master mix to minimize pipetting steps and eliminate tedious calculations, maximizing both convenience and accuracy. Only primers, template, and probes (QuantiTect Probe Kits only) need to be added to prepare the final amplification mix.

Master mixes for QuantiTect kits can be stored at 2–8°C, allowing even faster setup of amplification reactions by eliminating thawing time.

Applications

QuantiTect PCR Kits (for PCR and two-step RT-PCR) and QuantiTect RT-PCR Kits (for one-step RT-PCR) provide accurate real-time quantification of DNA, cDNA, and RNA targets on any real-time PCR cycler, including:

Highly Sensitive Quantification Using the QuantiTect Probe RT-PCR Kit

Amplification plots of real-time RT-PCR analysis using the ABI PRISM 7700 Sequence Detection System (Applied Biosystems). One-step, quantitative RT-PCR was carried out using the QuantiTect Probe RT-PCR Kit (QIAGEN) or one- step, quantitative RT-PCR kits from suppliers AII, L, or SIII, as indicated, according to suppliers’ instructions. Reactions were performed with the indicated number of copies of an in vitro transcript of the TATA-box binding protein (TBP). Inset: Agarose-gel analyses of end-point PCR results. M: markers.