Rotor-Gene Multiplex RT-PCR Kit

For ultrafast, multiplex, one-step qRT-PCR gene expression analysis on Rotor-Gene cyclers

Features

Optimized for ultrafast, reliable results on Rotor-Gene cyclers
Sensitive detection of multiple RNA targets in 1 tube
Successful multiplex PCR without the need for optimization
Efficient coamplification of low- and high-abundance targets

Product Details

The Rotor-Gene Multiplex RT-PCR Kit is designed for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly reliable quantification in multiplex, real-time one-step RT-PCR using sequence-specific probes. Depending on the cycler configuration, up to 4 RNA targets (e.g., 1 control gene and 3 target genes) can be quantified simultaneously in the same tube. Outstanding performance is achieved through the combination of a specially optimized master mix and the unique Rotor-Gene cycler. For convenience, the master mix can be stored at 2–8°C.

IMPORTANT NOTE: As announced earlier, the production of the Rotor-Gene kits has been discontinued since mid-2021. Hence, these products will be available only until stocks last. Visit the product page of the successor kit to view improved features or to request a trial kit.

For more information and FAQs on this transition, visit: www.qiagen.com/PCRresource.

Performance

The Rotor-Gene Multiplex RT-PCR Kit reliably quantifies low- to high-abundance targets in duplex, triplex, and 4-plex assays from as little as 10 pg template, and can detect 10 copies of target (see figures "" and ""). All targets in a multiplex assay are amplified with equally high PCR efficiency (see figure ""). This enables reliable relative quantification, where the expression of a target gene is normalized to that of a control gene.

See figures

Reliable duplex analysis.

Efficient, sensitive duplex analysis.

Highly efficient 4-plex analysis.

Principle

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 Rotor-Gene Multiplex RT-PCR Kit enables reliable multiplex quantification of RNA targets on the Rotor-Gene Q without the need for optimization of reaction and cycling conditions (see flowchart " QIAGEN multiplex kits"). Real-time one-step RT-PCR is carried out, enabling reverse transcription and PCR to take place sequentially in the same reaction vessel. Since it is not necessary to transfer the finished reverse transcription reaction to another tube for PCR, the real-time RT-PCR procedure is streamlined, making high-throughput analysis possible.

Highly specific amplification is assured through a balanced combination of K⁺ and NH₄⁺ ions, which promote specific primer annealing and enable high PCR specificity and sensitivity, while synthetic Factor MP, an innovative PCR additive specially developed for challenging multiplex PCR applications, allows different amplicons in the same reaction to all be amplified with the same high efficiency (see figure " Unique PCR buffer").

Fast cycling without compromising performance is achieved using Q-Bond, a novel PCR additive that considerably shortens cycler run times (see figure " Fast primer annealing"). In addition, an optimized mix of reverse transcriptases provides efficient cDNA synthesis in just 15 minutes, while the highly stringent hot-start enzyme HotStarTaq Plus DNA Polymerase is rapidly activated at the start of PCR by a brief 5-minute incubation at 95ºC.

Components of 2x Rotor-Gene Multiplex RT-PCR Kit*
Component	Features	Benefits
HotStarTaq Plus DNA Polymerase	5 min activation at 95ºC	Set up of qPCR reactions at room temperature
Rotor-Gene Multiplex RT-PCR Buffer	Balanced combination of NH₄⁺ and K⁺ ions	Specific primer annealing ensures reliable qPCR results
	Synthetic Factor MP	Reliable multiplexing analysis of up to 4 genes in the same tube
	Unique Q-Bond additive	Faster PCR run times enable faster results and more reactions per day
Rotor-Gene RT Mix	Special blend of reverse transcriptases with a high affinity for RNA	RNA can be transcribed in just 15 minutes, even through complex secondary structures

See figures

QIAGEN multiplex kits.

Unique PCR buffer.

Fast primer annealing.

Procedure

A ready-to-use master mix eliminates the need for optimization of reaction and cycling conditions, such as primer and probe concentrations. Simply add template RNA, primer–probe sets, and the supplied reverse transcriptase mix to the master mix and program the cycler. The handbook supplied with the kit lists recommended dyes and contains a single protocol for all multiplex RT-PCR assays.

Applications

The Rotor-Gene Multiplex RT-PCR Kit is optimized for fast, real-time one-step RT-PCR analysis using sequence-specific probes on the Rotor-Gene Q. It is also compatible with the Rotor-Gene 3000 and the Rotor-Gene 6000. Up to 4 RNA targets can be simultaneously and rapidly quantified in the same tube, increasing throughput and saving precious sample material.

Supporting data and figures

Reliable duplex analysis.

Duplex, real-time one-step RT-PCR was performed using the Rotor-Gene Multiplex RT-PCR Kit and self-designed TaqMan assays for 28S rRNA (Cyanine 670 dye; data in inset) and PPIA (cyclophilin A, FAM dye). Triplicate reactions were run on the Rotor-Gene 3000 using leukocyte RNA as template (10-fold dilutions from 100 ng to 10 pg). The amplification plots for the duplex reactions (colored) overlapped with those for control singleplex reactions (gray), demonstrating the reliability of the duplex analysis.

Specifications

Features	Specifications
Applications
Real-time or endpoint
Single or multiplex
With or without ROX
SYBR Green I or sequence-specific probes
Reaction type
Sample/target type
Thermal cycler

Resources

FAQ

Yes, please visit our website section 'Using endogenous control genes in real-time RT-PCR' for general information. It provides a list of relative gene expression levels for commonly used human and mouse reference genes.

We offer a set of ready-to-order control genes for use in SYBR Green based as well as probe based real-time RT-PCR.

QuantiTect Primer Assays are primer sets for use in SYBR Green based real-time RT-PCR analysis.
QuantiFast Probe Assays are primer-probe sets for use in dual-labeled probe based real-time RT-PCR.

In addition, you may want to refer to the following citations on reference gene selection for quantitative real-time PCR:

• Vandesompele J, De Preter K, Pattyn F, Poppe B, Van Roy N, DePaepe A, Speleman F [2002]: Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol 2002, 3:0034.

• Radonic A, Thulke S, Mackay IM, Landt O, Siegert W, Nitsche A., 2004. Guideline to reference gene selection for quantitative real-time PCR. Biochem Biophys Res Commun. 313(4): 856-62.

• Katrien Smits,Karen Goossens, Ann Van Soom, Jan Govaere, Maarten Hoogewijs, Emilie Vanhaesebrouck,Cesare Galli, Silvia Colleoni, Jo Vandesompele, and Luc Peelman [2009]Selection of reference genes for quantitative real-time PCR in equine in vivo and fresh and frozen-thawed in vitro blastocysts. BMC Res Notes. Dec 11;2:246.

FAQ ID -2371

The most important prerequisite for any gene expression analysis experiment is the preparation of consistently high-quality RNA from every experimental sample. Contamination by DNA, protein, polysaccharide, or organic solvents can jeopardize the success of an experiment.

Genomic DNA contamination in an RNA sample compromises the quality of gene expression analysis results. The contaminating DNA inflates the OD reading of the RNA concentration. It is also a source of false positive signals in RT-PCR experiments.

RNase contamination degrades RNA samples whichcauses low signal and false-negative results in PCR.

Residual polysaccharides, collagen, other macromolecules, and organic solvents in an RNA sample can inhibit the activity of DNase, which may interfere with DNase treatment for genomic DNA removal. These contaminants may also inhibit reverse transcriptase and DNA polymerase, leading to lower reverse transcription efficiency and reduced PCR sensitivity.

For fast purification of high-quality RNA we recommend QIAGEN’s RNeasy Kits like the RNeasy Mini Kit, the RNeasy Plus Universal Kit, or the RNeasy FFPE Kit.

FAQ ID -2655

The Ct or threshold cycle value is the cycle number at which the fluorescence generated within a reaction crosses the fluorescence threshold, a fluorescent signal significantly above the background fluorescence. At the threshold cycle, a detectable amount of amplicon product has been generated during the early exponential phase of the reaction. The threshold cycle is inversely proportional to the original relative expression level of the gene of interest.

FAQ ID -2682

Our unique multiplex PCR buffer system with ammonium and potassium ions and Factor MP has been further optimized in QuantiFast and Rotor-Gene Kits. We have also discovered Q-Bond, a buffer component which supports the rapid formation of the polymerase–primer–template complex, leading to reduced annealing times.

FAQ ID -1430

The Rotor-Gene and QuantiFast Multiplex RT-PCR Kits allow reliable detection down to 10 target copies. Detection of lower copy numbers down to single copy level may also be possible; however, this depends on the stochastics when working with highly diluted samples. Additional optimization of primer/probe design is usually required.

FAQ ID -2144

The specific features of Rotor-Gene Kits and Rotor-Gene cyclers work synergetically to enable an ultrafast-cycling protocol. We do not guarantee that the performance of the Rotor-Gene Multiplex RT-PCR Kit with the same cycling protocol will be the same on other cyclers.

FAQ ID -2142

Check the template quality and integrity by amplifying an endogenous control gene. Check the amplicon by QIAxcel Advanced system or agarose gel electrophoresis to show that amplification was successful.

Determine whether the gene of interest is expressed in your sample. See How can I find out if my gene of interest is express in a specific tissue type or cell line. Ensure the assay setup and cycling conditions are correct, and that the data collection channel matches the emission wavelength of the fluorescent dye used. Use a control sample in which the gene of interest is definitely expressed.

If the issue persists, please send the original run file to QIAGEN Technical Services.

FAQ ID -9091

No. UNG treatment does not provide any advantage for the QuantiFast and Rotor-Gene PCR kits because the mastermixes do not contain dUTP. Use the QuantiTect kits if you intend to use the UNG treatment.

FAQ ID -9092

Ensure PCR amplicons are as short as possible, ideally 60–150 bp. Always use the same algorithm or software to design the primers and probes. For optimal results, only combine assays that have been designed using the same parameters.

Check the functionality of each set of primers and probes in individual assays before combining the different sets in the multiplex assay. Choose compatible reporters and quenchers based on a specific instrument. See How do I select appropriate reporter and quencher combinations for multiplex PCR.

FAQ ID -9093

For duplex analysis, using non-fluorescent quenchers (e.g., Black Hole Quencher^®) is preferred over fluorescent quenchers (e.g., TAMRA fluorescent dye). For triplex and 4-plex analysis, QIAGEN strongly recommends using non-fluorescent quenchers. Generally, use the green channel, the yellow channel, and the orange and crimson channels to detect the least abundant target, the second least abundant target, and the two most abundant targets, respectively. For instrument-specific recommendations, please see the handbooks for the QuantiTect Multiplex PCR kit, QuantiFast Multiplex kit or Rotor-Gene Multiplex kit.

FAQ ID -9094

The REST 2009 (Relative Expression Software Tool) software applies mathematic models that compensate for the different PCR efficiencies of the gene of interest and reference genes. In addition, the software can use multiple reference gene normalization to improve the reliability of result, as well as provides statistical information suitable for robust comparison of expression in groups of treated and untreated. QIAGEN offers the REST 2009 software free of charge.

FAQ ID -9095

Depending on primer design and copy number of target, primer-dimer may occur and its signal might be detected. Typical strategies against this are to optimize PCR conditions and/or redesign the assay.

Alternatively, an additional data-acquisition step can be added to the 3-step cycling protocol. First, determine the melting temperatures (T_m) for both the amplicon and the primer-dimer. Then, add a 15 second data-acquisition step with a temperature that is higher than the primer-dimer T_m, but approximately 3ºC lower than the specific amplicon T_m.

FAQ ID -9096

Reliable HRM analysis results depend on template quality, highly specific HRM PCR kit with a saturation dye, a real-time instrument with HRM capability, and powerful software package. Factors critical for successful HRM analysis are:

Use the same genomic DNA purification procedure for all samples being analyzed by HRM. This avoids variation due to differing composition of elution buffers.
DNA template concentrations should be normalized using the same dilution buffer. Ensure the C_T values are below 30 and differ no more than 3 C_T values across individual samples.
Design assays with amplicon length 70–350 bp. For SNP analysis, use amplicon length 70–150 bp.
Always start with 0.7 µM primer concentration

For more details, please refer to the HRM Technology – FAQs and the Critical Success Factor for HRM performance.

FAQ ID -9097

The gDNA wipeout buffer incubation step can be skipped when the total RNA is free from genomic DNA. However, the gDNA wipeout buffer is still required to be added because the reverse transcription step is optimized in the presence of components in the gDNA wipeout buffer.

FAQ ID -9098

If the extra peaks seem irregular or noisy, do not occur in all samples, and occur at temperatures less than 70 ºC, then these peaks may not represent real PCR products and instead may represent artifacts caused by instrument settings.

Usually extra peaks caused by secondary products are smooth and regular, occur reproducibly in most samples, and occur at temperatures greater than 70 ºC. Characterization of the product by agarose gel electrophoresis is the best way to distinguish between these cases. If only one band appears by agarose gel then the extra peaks in the dissociation curve are instrument artifacts and not real products. If this is the case, refer to the thermal cycler user manual, and confirm that all instrument settings (smooth factor, etc.) are set to their optimal values.

FAQ ID -90990

Store the standards at a high concentration in aliquots at -20^oC to -70^oC. If using low concentrations, stabilize standards with carrier nucleic acid. It is always best to use freshly diluted standards for each experiment. If possible, use siliconized tubes for standard (and target) dilutions. This will prevent any unspecific binding of nucleic acids to the plastic.

FAQ ID -9099