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Rotor-Gene Multiplex PCR Kit

用于在Rotor-Gene PCR仪上超快速的两步法多重qRT-PCR基因表达分析

Features

  • 在Rotor-Gene PCR仪上获得超快速、可靠的结果
  • 单管内灵敏检测多个靶序列
  • 表现优越的多重PCR,无需优化
  • 准确检测靶基因的微小差异

Product Details

Rotor-Gene Multiplex PCR Kit专用于Rotor-Gene Q实时荧光定量PCR分析仪和其他Rotor-Gene PCR仪,进行超快速、高度可靠的多重定量两步法RT-PCR,使用序列特异性探针。通过调整热循环仪,可在同一反应中同时定量分析多达4个cDNA靶基因(如1个对照基因和3个靶基因)。特殊优化的预混液配合独特的Rotor-Gene PCR仪,表现卓越。为方便使用,预混液可储存在2–8°C。

Performance

使用Rotor-Gene Multiplex RT-PCR Kit,多达4个cDNA靶基因可在单管内同步快速定量分析,提高通量的同时,节约珍贵的样本(参见 " Reliable multiplex analysis" and " Reliable duplex analysis")。多重分析中不同表达水平的靶基因在同一个反应管中以同样的效率扩增,可确保可靠的相对定量分析。
See figures

Principle

扩增参照和靶基因在同一个反应管中,减少操作误差,提高了基因定量分析的可靠性。Rotor-Gene Multiplex RT-PCR Kit确保在Rotor-Gene Q实时荧光定量PCR分析仪上进行可靠的多重cDNA靶基因定量分析,无需优化反应和循环条件(参见" QIAGEN multiplex kits")。 平衡的K+和NH4+离子组合确保高度特异性,促进特异性引物退火,PCR特异性高、灵敏度好。一种新颖的PCR添加剂合成的Factor MP专用于多重PCR应用,能够以同样的效率扩增在同一反应中不同的扩增子(参见 " Unique PCR buffer")。

一种新颖的PCR添加剂Q-Bond可缩短扩增时间,确保快速扩增的同时不影响表现(参见" Fast primer annealing")。 此外,高度严谨的热启动酶HotStarTaq Plus DNA Polymerase在95ºC条件下只需5分钟即可快速激活。

2x Rotor-Gene Multiplex PCR Kit*的组分
组分 特性 优势
HotStarTaq Plus DNA聚合酶 95ºC条件下5分钟激活 室温下建立qPCR反应体系
Rotor-Gene Multiplex PCR缓冲液 NH4+和K+平衡组合预混液 特异性引物退火,确保可靠的qPCR结果
合成的Factor MP 在同一反应管中可靠的进行多达4个基因的多重分析
独特的Q-Bond添加剂 更快速的PCR循环,快速获得结果,一天可进行更多的反应
*  包含dNTP混合液(dATP、dCTP、dGTP和dTTP)。
See figures

Procedure

应用即用型预混液无需优化反应和循环条件。只需在预混液中添加模板cDNA和引物探针对,然后设置PCR仪即可开始实验。试剂盒中提供的操作手册中列出了推荐染料,并为多重qPCR分析提供了单独的实验方案。

为了两步法定量RT-PCR获得更加优化的结果,推荐使用QuantiTect Reverse Transcription Kit合成cDNA。该试剂盒只需20分钟即可快速合成cDNA,并可完全去除基因组DNA的污染。

Applications

Rotor-Gene Multiplex PCR Kit专用于Rotor-Gene Q实时荧光定量PCR分析仪,使用序列特异性探针进行快速的两步法RT-PCR分析。该试剂盒也与Rotor-Gene 3000和Rotor-Gene 6000兼容。多达4个cDNA靶基因可在单管内同步快速定量分析,提高通量的同时,节约珍贵的样本。

Rotor-Gene Multiplex RT-PCR Kit用于Rotor-Gene PCR仪上,使用序列特异性探针对RNA靶分子进行超快速的一步法qRT-PCR多重分析。

Supporting data and figures

Specifications

FeaturesSpecifications
ApplicationsReal-time quantification of genomic DNA or cDNA targets in a multiplex format
Real-time or endpointReal-time
Reaction typeReal-time PCR and two-step RT-PCR
Sample/target typeDNA, cDNA
DescriptionFor ultrafast quantitative multiplex real-time PCR and two-step RT-PCR using sequence-specific probes
Single or multiplexMultiplex
SYBR Green I or sequence-specific probesSequence-specific probes
Thermal cyclerRotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000
With or without ROXWithout ROX dye

Resources

FAQ

Do you have any information or guidelines regarding the choice of reference genes for real-time PCR?

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.

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
Do the master mixes in Rotor-Gene Kits contain dUTP to allow UNG pretreatment?

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
If housekeeping and target genes differ significantly in abundance, will they both be amplified with equal efficiency using the Rotor-Gene Multiplex PCR Kit?

Yes. The optimized master mix of the Rotor-Gene Multiplex PCR Kit ensures that all gene targets in a multiplex reaction are amplified with the same efficiency and sensitivity as in corresponding singleplex reactions, independent of starting copy numbers.

 

FAQ ID -2129
Are probes other than TaqMan® probes, such as FRET probes, compatible with fast cycling with the Rotor-Gene Multiplex PCR Kit?

The Rotor-Gene Multiplex PCR Kit has been developed primarily for use with TaqMan® probes. Protocols for probes other than TaqMan® probes have not been tested.

See trademarks

FAQ ID -2130
Why is the initial activation step different for Rotor-Gene Probe, SYBR Green and Multiplex Kits?

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
How do you achieve fast cycling, yet still deliver the same performance in PCR as that achieved with standard cycling?

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
Do limiting primer concentrations need to be determined when using the Rotor-Gene Multiplex PCR Kit?

No, that is not necessary. Simply use the primer concentrations specified in the protocols in the handbook supplied with your Rotor-Gene Multiplex PCR Kit.

 

 

FAQ ID -2128
What real-time cycler should I use for my qPCR experiments?
There are several manufacturers of high-quality real-time cyclers. These include QIAGEN's Rotor-Gene Q, Applied Biosystems, BioRad, Stratagene, Eppendorf, Roche, TaKaRa, Fluidigm, and Cepheid. The important thing to keep in mind is that, once you select an instrument to use, you must use compatible Rotor-Gene Discs and tubes, 96 or 384 well plates, and qPCR master mixes that are optimized for use in that particular instrument.  For example, QIAGEN's Rotor-Gene SYBR Green, Probe, and Multiplex real-time master mixes.  
FAQ ID -2670
Are Rotor-Gene Kits compatible with reaction setup using the QIAgility instrument?

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
Are TaqMan® Gene Expression Assays from Applied Biosystems compatible with the Rotor-Gene Multiplex PCR Kit?

Yes, simply use the assays at a final concentration of 1x with the Rotor-Gene Multiplex PCR Kit.

See trademarks

FAQ ID -2131
What are the main differences between Rotor-Gene and QuantiTect or QuantiFast PCR Kits?

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
Will Rotor-Gene Kits also work on the Rotor-Gene 6000 and 3000 cyclers?

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
What do I do if no fluorescent signal is detected in a real-time PCR assay?

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
Can I use uracil-N-glycosylase (UNG) with the QuantiFast and Rotor-Gene PCR kits?

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
How do I setup and validate a multiplex PCR assay with QIAGEN PCR kits?

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
How do I select appropriate reporter and quencher combinations for multiplex PCR?

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
How do I quantify gene expression levels if the amplification efficiencies are different between the genes of interest and endogenous reference gene?

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
How do I avoid collecting a fluorescence reading from primer-dimer with the QuantiTect SYBR Green PCR Kit?

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 (Tm) 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 Tm, but approximately 3ºC lower than the specific amplicon Tm.

FAQ ID -9096
How do I ensure reliable results for High Resolution Melting (HRM) assays?

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 CT values are below 30 and differ no more than 3 CT 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
Can I skip the gDNA wipeout buffer treatment step for the QuantiTect Reverse Transcription Kit?

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
Why do I see multiple high-intensity peaks in my qPCR dissociation curve at temperatures less than 70ºC?

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
How should I handle and store absolute quantitation standards for real-time experiments?
Store the standards at a high concentration in aliquots at -20oC to -70oC. 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
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