For multiplex, one-step qRT-PCR using sequence-specific probes for gene expression analysis
Multiplex analysis with no need for optimization
Analysis of multiple targets in a single reaction
Sensitive detection of as few as 10 copies of each target
Reliable quantification of target and reference genes
Detection of reference gene and up to 3 targets in the same tube
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.
For 1000 x 50 µl reactions: 25 ml 2x QuantiTect Multiplex RT-PCR NoROX Master Mix (without ROX dye), 0.5 ml QuantiTect Multiplex RT Mix, 20 ml RNase-Free Water
Comparable amplification in triplex PCR and singleplex PCRs.
Triplex, real-time one-step RT-PCR was performed on the Applied Biosystems 7500 using the QuantiTect Multiplex RT-PCR Kit and TaqMan probes. The template was 20 ng total RNA from the Burkitt's lymphoma cell line Ramos. Reactions were performed in triplicate. 28S rRNA was detected using a HEX labeled probe. POLD3 (accessory subunit of DNA polymerase delta 3) was detected using a FAM labeled probe. CDK2 (cell cycle-dependent kinase 2) was detected using a Cy5 labeled probe. For comparison, the targets were also quantified by singleplex, real-time one-step RT-PCR (black curves). Curves for triplex PCR and singleplex PCRs overlap, demonstrating comparable amplification (i.e., equivalent CT values).
Detection of down to 10 copies of target RNA in duplex PCR.
Duplex, real-time one-step RT-PCR was performed on the Applied Biosystems 7500 using the QuantiTect Multiplex RT-PCR Kit and TaqMan probes. The target was [A] 1000, 100, or 10 copies of an in vitro transcript of a viral gene, each spiked with [B] 1000 copies of synthetic internal control RNA. Reactions were performed in replicate. Target RNA was detected using a FAM labeled probe. Internal control was detected using a HEX labeled probe. 7 replicates of 10 copies of target RNA were reproducibly detected.
Comparable amplification in 4-plex PCR and singleplex PCRs.
4-plex, real-time one-step RT-PCR was performed on the LightCycler 2.0 using the QuantiTect Multiplex RT-PCR NR Kit and TaqMan probes. The template was 10, 1, or 0.1 ng of total RNA purified from K562 cells. [A] c-myc (a proto-oncogene) was detected using a FAM labeled probe. [B] GAPDH (glyceraldehyde 3-phosphate dehydrogenase) was detected using a HEX labeled probe. [C] HSP89 (a heat shock protein) was detected using a Texas Red labeled probe. [D] H28S (28S rRNA) was detected using an Alexa Fluor 660 labeled probe. For comparison, the targets were also quantified by singleplex, real-time one-step RT-PCR (black curves). Curves for 4-plex and singleplex PCRs overlap, showing comparable amplification (i.e., equivalent CT values).
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.
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.
Components of 2x QuantiTect Multiplex RT-PCR Kit
HotStarTaq DNA Polymerase
15 min activation at 95ºC
Set-up of qPCR reactions at room temperature
QuantiTect Multiplex RT-PCR Buffer
Balanced combination of NH4+ and K+ ions
Specific primer annealing ensures reliable PCR results
Synthetic Factor MP
Reliable multiplexing analysis of up to 4 genes in the same tube
Includes dUTP, which partially replaces dTTP and enables optional UNG treatment of reactions
Eliminates contamination from carryover of PCR products by optional UNG treatment
For normalization of fluorescent signals on Applied Biosystems and, optionally, Agilent instruments
Precise quantification on cyclers that require ROX dye. Does not interfere with reactions on other real-time cyclers
Omniscript and Sensiscript Reverse Transcriptases
Special blend of enzymes with high affinity for RNA
RNA can be transcribed, even through complex secondary structures
*ROX dye is either present in the master mix or not. See table "Choosing the right QuantiTect Multiplex RT-PCR Kit".
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.
Choosing the right QuantiTect Multiplex RT-PCR Kit
Supplied in master mix
QuantiTect Multiplex RT-PCR Kit
Cyclers from Applied Biosystems
Absent from master mix
QuantiTect Multiplex RT-PCR NR Kit
Rotor-Gene cyclers, and cyclers from Bio-Rad, Cepheid, Eppendorf, Roche, Agilent, and other suppliers
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.
Real-time quantification of RNA targets in a multiplex format
For quantitative, multiplex, real-time one-step RT-PCR using sequence-specific probes
Real-time one-step RT-PCR
Real-time or endpoint
Single or multiplex
SYBR Green I or sequence-specific probes
Real-time cyclers dedicated for multiplex PCR (e.g., most Applied Biosystems real-time cyclers, Roche LightCycler 480, and Bio-Rad iCycler iQ)
For 100 x 50 µl reactions: QIAGEN OneStep RT-PCR Enzyme Mix (1 x 200 µl), 5x QIAGEN OneStep RT-PCR Buffer (1 x 1 ml), dNTP Mix (1 x 200 µl, 10 mM each), 5x Q-Solution (1 x 2 ml), RNase-Free Water (2 x 1.9 ml)