QuantiTect Multiplex PCR Kits
For multiplex, real-time PCR and RT-PCR using sequence-specific probes
QuantiTect Multiplex PCR Kits enable reliable quantification of up to 5 gDNA or cDNA targets in a single tube by multiplex, real-time PCR or two-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 qPCR 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 PCR Kit for cyclers that require ROX dye for fluorescence normalization, and the QuantiTect Multiplex PCR NoROX Kit for all other cyclers. For convenience, the master mix can be stored at 2–8°C.
The QuantiTect Multiplex PCR Master Mix ensures that the PCR products in a multiplex reaction are amplified with the same efficiency and sensitivity as the PCR products in the corresponding single-amplification reactions (see table “Comparable threshold cycle (CΤ) values with triplex and singleplex PCRs”, and figures “Comparable results with 4-plex and singleplex PCRs” and "Equivalent CT values in triplex PCR and singleplex PCR").
As few as 10 copies of a target gene can be detected with the kits, even if the copy number of the reference gene in the same reaction is up to 106-fold greater (see figures “Detection of 10 copies of target gene with excess reference gene", "Successful quantitative triplex qPCR of low and high abundance targets" and the corresponding table "Successful quantitative triplex qPCR of low and high abundance targets").
QuantiTect Multiplex PCR Kits enable success in multiplex, two-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 PCR Master Mix contains a balanced combination of K+ and NH4+ ions as well as the 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, HotStarTaq DNA Polymerase provides a stringent hot start, preventing the formation of nonspecific products.
The QuantiTect Multiplex 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.
QuantiTect Multiplex 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.
For optimal results in real-time two-step RT-PCR, we recommend synthesizing cDNA using the QuantiTect Reverse Transcription Kit. The kit provides fast cDNA synthesis in just 20 minutes with integrated removal of genomic DNA contamination.
QuantiTect Multiplex PCR Kits can be used for gene expression analysis of cDNA or quantification of gDNA 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 PCR Kit, which has been specially developed for fast cycling on these instruments.
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