Rotor-Gene Probe RT-PCR Kit

For ultrafast, one-step qRT-PCR using sequence-specific probes on Rotor-Gene cyclers

Features

Optimized for ultrafast, reliable results on Rotor-Gene cyclers
Sensitive detection of even low copy numbers
Accurate detection of a wide range of template amounts
Specially formulated, ready-to-use master mix for fast cycling

Product Details

The Rotor-Gene Probe RT-PCR Kit is designed for use with the Rotor-Gene Q and other Rotor-Gene cyclers, providing ultrafast, highly sensitive quantification of RNA targets with real-time one-step RT-PCR using sequence-specific probes. 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

With the Rotor-Gene Probe RT-PCR Kit, low-abundance transcripts are reliably quantified (see figure "").

See figures

Sensitive detection using a sequence-specific probe.

Principle

The Rotor-Gene Probe RT-PCR Kit enables reliable quantification of RNA templates on the Rotor-Gene Q without the need for optimization of reaction and cycling conditions. The kit is designed for use with hydrolysis probes (e.g., TaqMan® probes). RNA is used as the template in a reaction where reverse transcription and PCR take place sequentially in the same reaction vessel. Since it is not necessary to transfer the finished RT 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, enabling high PCR specificity and sensitivity (see figure " Specific primer annealing"). 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").

Components of 2x Rotor-Gene Probe 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 Probe RT-PCR Buffer	Balanced combination of NH₄⁺ and K⁺ ions	Specific primer annealing ensures reliable qPCR results
Rotor-Gene Probe RT-PCR Buffer	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 10 minutes, even through complex secondary structures

See figures

Specific primer annealing.

Fast primer annealing.

Procedure

The ready-to-use Rotor-Gene Probe RT-PCR Master Mix eliminates the need for optimization of reaction and cycling conditions. Just add template RNA, primers, probe, and the supplied reverse transcriptase mix to the master mix and program the cycler. Instructions are provided in the detailed handbook supplied with the kit.

Hydrolysis probes (e.g., TaqMan® probes) can be used in combination with the Rotor-Gene Probe RT-PCR Kit on the Rotor-Gene Q for fast and sensitive quantification — simply add the primer-probe mix and template to the master mix.

Applications

The Rotor-Gene Probe RT-PCR Kit is well suited for use in fast, real-time one-step RT-PCR of RNA targets using sequence specific probes on the Rotor-Gene Q. It is also compatible with Rotor-Gene 3000 and Rotor-Gene 6000 cyclers.

Supporting data and figures

Sensitive detection using a sequence-specific probe.

Tenfold dilutions of human leukocyte RNA (100 ng to 1 ng) were used as template in real-time one-step RT-PCR. Triplicate reactions were run using a TaqMan Gene Expression Assay for IL12RB1 (interleukin 12 receptor, beta 1). Greater sensitivity (i.e., lower C_T values) was achieved with [A] the Rotor-Gene Q and Rotor-Gene Probe RT-PCR Kit than with [B] an instrument and kit from Supplier A_II.

Specifications

Features	Specifications
Applications	Real-time quantification of RNA targets
Reaction type	Real-time one-step RT-PCR
Real-time or endpoint	Real-time
Thermal cycler	Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000
SYBR Green I or sequence-specific probes	Sequence-specific probes
Sample/target type	RNA
Single or multiplex	Single
Description	For ultrafast quantitative real-time one-step RT-PCR using sequence-specific probes
With or without ROX	Without ROX dye

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

No. The master mixes in Rotor-Gene Kits contain dTTP instead of dUTP. If UNG treatment is required, we recommend using QuantiTect +UNG Kits. QuantiTect Kits are also compatible with the Rotor-Gene Q; however, the kits require a significantly longer cycling time.

FAQ ID -2117

The buffer composition, which affects the initial reactivation of HotStarTaq Plus DNA Polymerase, has been optimized for each respective Rotor-Gene Kit.

FAQ ID -2118

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 majority of the Rotor-Gene Kit data shown in our literature has been generated with the help of the QIAgility instrument. We did not observe any problems during the pipetting steps.

FAQ ID -2116

Rotor-Gene Kits are specifically developed for the Rotor-Gene Q PCR Cycler. The unique rotary system of the cycler combined with the kits’ proprietary buffer system enable ultrafast cycling. Rotor-Gene Kits do not contain ROX dye since no normalization to a passive reference is required. Also, Rotor-Gene Kits do not contain dUTP; therefore, UNG pretreatment is not possible.

FAQ ID -2119

Yes. Rotor-Gene Kits will also work on the Rotor-Gene 6000 and Rotor-Gene 3000 PCR cyclers with the cycling conditions specified in the Rotor-Gene kit handbooks.

FAQ ID -2121

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