QuantiTect Multiplex RT-PCR Kits
For multiplex, one-step qRT-PCR using sequence-specific probes for gene expression analysis
QuantiTect Multiplex RT-PCR Kits enable 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 and reliable multiplex qRT-PCR on any real-time cycler without the need for optimization. The dNTP mix includes dUTP, allowing optional treatment with UNG. Two kit formats are available: 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. For convenience, the master mix can be stored at 2–8°C.
QuantiTect Multiplex RT-PCR Kits are intended for molecular biology applications. These products are not intended for the diagnosis, prevention, or treatment of a disease.
Performance
QuantiTect Multiplex RT-PCR Kits provide sensitive detection of as little as 10 copies of target sequence (see figure "Detection of down to 10 copies of target RNA in duplex PCR"). Optimal results are achieved even in 4-plex assays to quantify transcripts that strongly differ by up to 100,000-fold in starting copy number (see figure "Comparable amplification in 4-plex PCR and singleplex PCRs"). 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.
Principle
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. QuantiTect Multiplex RT-PCR Kits enable success in multiplex, one-step RT-PCR on the first attempt (see flowchart "QIAGEN multiplex kits"). The optimized master mix 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. As few as 10 copies of a target gene can be detected with the kit. Amplifying reference and target genes in the same reaction instead of in separate reactions increases the reliability of gene quantification by minimizing handling errors. The QuantiTect Multiplex RT-PCR Master Mix contains a balanced combination of K+ and NH4+ ions as well as unique synthetic Factor MP, which together promote stable and efficient annealing of primers and probes to the nucleic acid template, enabling high PCR efficiency (see figure "Unique PCR buffer"). 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. The QuantiTect Multiplex 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
QuantiTect Multiplex RT-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. If required, reactions can be pretreated with uracil-N-glycosylase (UNG; not supplied) to eliminate carryover of PCR products from previous reactions. Kits are available with or without ROX passive reference dye in the master mix, enabling use on virtually any real-time cycler (see table). Due to the optimized ROX concentrations, detection of even low copy numbers is achieved through automatic data analysis.
Applications
QuantiTect Multiplex 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 Multiplex RT-PCR Kit, which has been specially developed for fast cycling on these instruments.
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