Rotor-Gene Multiplex RT-PCR Kit

用于在Rotor-Gene PCR仪上超快速的多重一步法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
单管内高灵敏检测多个RNA靶序列
表现优越的多重PCR，无需优化
低丰度和高丰度靶基因高效共扩增

Product Details

Rotor-Gene Multiplex RT-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，可超快速进行基于序列特异性探针的可靠的多重一步法real-time RT-PCR定量分析。通过调整PCR仪，可在单管内同步定量分析多至4个RNA靶基因（如1个参照和3个靶基因）。配合经优化的预混液和具有独特转子设计的Rotor-Gene PCR仪共同使用，表现卓越。为便于使用，预混液应储存在2–8°C。

Performance

Rotor-Gene Multiplex RT-PCR Kit能够对低至高丰度靶基因可靠进行二重、三重、四重定量检测，可检测低至10 pg的模板及10个拷贝的靶基因（参见""和""）。多重分析中不同的靶基因以同样的高效率得以扩增（参见""）。因此可实现可靠的相对定量检测，标准品为对照基因。

See figures

Reliable duplex analysis.

Efficient, sensitive duplex analysis.

Highly efficient 4-plex analysis.

Principle

在同一反应管中扩增参照和靶基因，减少了手动操作失误，提高了基因定量检测的可靠性。Rotor-Gene Multiplex RT-PCR Kit能够在Rotor-Gene Q实时荧光定量PCR分析仪上可靠的多重定量检测RNA靶基因，无需优化反应条件（参见" QIAGEN multiplex kits"）。进行一步法real-time RT-PCR，使逆转录和PCR扩增在同一反应容器中相继进行。因为无需将逆转录产物转移到另一管中进行PCR，所以real-time RT-PCR的流程得以简化，可实现高通量分析。

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，逆转录酶在15分钟内即可高效的合成cDNA，而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 RT-PCR Kit组分*
组分	特点	优势
HotStarTaq Plus DNA Polymerase	95ºC条件下5分钟激活	室温下建立qPCR反应体系
Rotor-Gene Multiplex RT-PCR Buffer	浓度平衡的NH₄⁺和K⁺离子	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
Rotor-Gene RT Mix	RNA亲和性高的逆转录酶	RNA逆转录可在15分钟内完成，甚至包括复杂的二级结构

See figures

QIAGEN multiplex kits.

Unique PCR buffer.

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RNA和引物探针对、逆转录酶，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RT-PCR分析提供了单独的实验方案。

Applications

Rotor-Gene Multiplex RT-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一步法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。

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 quantification of RNA targets in a multiplex format
Real-time or endpoint	Real-time
Single or multiplex	Multiplex
With or without ROX	Without ROX dye
SYBR Green I or sequence-specific probes	Sequence-specific probes
Reaction type	Real-time one-step RT-PCR
Sample/target type	RNA
Thermal cycler	Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000

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