( ...)Custom and library siRNA for efficient gene silencing

High-quality short interfering RNA (siRNA) from QIAGEN allows efficient gene silencing in eukaryotic cell culture by targeting complementary endogenous mRNA. This effect, termed RNA interference (RNAi), enables targeted gene silencing in functional genomics studies. Now, QIAGEN offers a custom siRNA oligonucleotide synthesis service that includes a range of modifications and also library siRNAs directed against common target genes.

QIAGEN siRNA Oligo Synthesis Service offers:

• High-purity siRNA for efficient gene silencing
  
• Custom and library siRNA ready to use in cell transfection
  
• Expert advice on siRNA design
  
• Labeled siRNA

Multiplex PCR that simply works the new QIAGEN Multiplex PCR Kit

The new QIAGEN Multiplex PCR Kit is the first kit specifically developed for multiplex PCR. The simple multiplex master-mix solution eliminates the need for lengthy optimization procedures, such as adjusting the amounts of Mg2+ and enzyme or even, as frequently required, adjusting primer concentrations. Now standard multiplex PCR applications are fast and easy to perform.

Advantages of the new QIAGEN Multiplex PCR Kit:

No optimization required easy assay development with simple master mix
High specificity and sensitivity stringent hot start with HotStarTaq DNA Polymerase and increased sensitivity with the unique new multiplex PCR buffer
Versatile for many applications including typing of transgenic animals and plants, detection of bacteria and viruses, and microsatellite analyses
Easy to use and cost-effective simple reaction setup for fast and reproducible results

No optimization required

Establishing multiplex PCR assays is a tedious and time-consuming procedure. In most cases, not all PCR products are generated, and nonspecific background products, such as primer-dimers, are often observed. To get acceptable results requires extensive optimization of the amounts of Taq DNA polymerase, Mg2+, additional reagents, and primers. Often, cycling parameters or reaction buffer formulations need to be changed. In many cases, the results are still disappointing, requiring further extensive optimization (see flowchart). The QIAGEN Multiplex PCR Kit eliminates the need for optimization, making the development of multiplex PCR assays both simple and fast. The master mix contains pre-optimized concentrations of HotStarTaq DNA Polymerase and MgCl2, plus dNTPs and a PCR buffer newly developed for multiplex reactions. Excellent results can be achieved the first time using fixed primer concentrations and the standard multiplex PCR cycling protocol, included in the QIAGEN Multiplex PCR Handbook. Typically, there is no need to adjust reaction parameters (see flowchart).

Multiplex PCR Setup

High specificity and sensitivity

Nonspecific PCR products and primer-dimers are often generated during multiplex PCR due to the large amount of different primers in the reaction. Amplification of these nonspecific products competes with amplification of the desired PCR products, leading to decreased sensitivity. With the QIAGEN Multiplex PCR Kit, specificity is significantly increased by use of HotStarTaq DNA Polymerase in the master mix. This enzyme enables a highly stringent hot start and allows reaction setup at room temperature without nonspecific artifacts. The newly developed multiplex PCR buffer in the master mix further enhances the sensitivity of the multiplex reaction. This innovative, new formulation was specifically developed and optimized for multiplex PCR. In contrast to conventional PCR reagents, the new buffer contains a balanced combination of salts and additives to ensure comparable efficiencies for annealing and extension of all primers in the reaction (Figure 1).

Successful 16plex PCR Using the QIAGEN Multiplex PCR Kit

Figure 1 Multiplex PCR of 16 targets (99C955 bp) was carried out for 35 cycles using standard conditions (Std) for the QIAGEN Multiplex PCR Kit (Q) without further optimization or using a variety of conditions with a hot-start DNA polymerase from Supplier AII (AII). A Comparison using 2.5 units/50 l reaction of the hot-start DNA polymerase from Supplier AII and with the indicated Mg2+ concentrations. B Comparison using the optimized Mg2+ concentration (3.5 mM) for the hot-start DNA polymerase from Supplier AII and the indicated amounts of enzyme per 50 l reaction. M: markers.

Versatile for many applications

The QIAGEN Multiplex PCR Kit is well suited for many types of multiplex PCR applications using different detection methods. PCR products can be visualized on agarose gels or detected using fluorescently labeled primers. Detection of fluorescently labeled products can be performed on automatic gel-based DNA sequencers (e.g., the ABI PRISM 377 DNA Sequencer; Figure 2), as well as those based on capillary electrophoresis (e.g., the ABI PRISM 310 Genetic Analyzer). The QIAGEN Multiplex PCR Kit has been successfully used for typing and analysis of transgenic animals and plants, amplification of multiple DNA regions for SNP analysis, and typing and detection of bacteria and viruses. A special protocol for amplification and analysis of microsatellites is also provided. In addition, the kit contains a protocol for multiplex amplification using Q-Solution, suitable for targets with high GC content or complex secondary structure.

Microsatellite Analysis with Optimized QIAGEN Protocol
Figure 2 Analysis of microsatellite loci D3S1358, TH01, D21S11, D18S51, and Penta E was carried out using 1 ng of K562 human genomic DNA and fluorescein-labeled primers. Reactions were analyzed on the ABI PRISM 377 Sequencer. Top: High sensitivity and uniform signal intensity using the QIAGEN Multiplex PCR Kit and the microsatellite protocol included in the QIAGEN Multiplex PCR Handbook. Bottom: Results using a hot-start DNA polymerase from Supplier AII.

Easy to use

The QIAGEN Multiplex PCR Kit contains a master mix specifically designed for multiplex PCR applications. The master-mix format makes handling and reaction setup both fast and easy. Use of a master-mix format reduces time and handling for reaction setup and increases reproducibility by eliminating many possible sources of pipetting errors. The master mix is stable at 20C and 4C for many months. Storage at 4C eliminates thawing time, providing even faster setup of multiplex PCR assays. Reaction setup at room temperature also makes the QIAGEN Multiplex PCR Kit the ideal tool for automated PCR setup.

The easy-to-follow protocol and the robust universal multiplex cycling conditions give you reliable results faster. This is especially important for simultaneous analysis of multiple samples, such as for genotyping transgenic animals and plants (Figure 3).

Fast and easy assay development withincreased reproducibility makes the QIAGEN Multiplex PCR Kit a cost-effective and timesaving solution for multiplex PCR applications.

Genotyping Transgenic Mice
Figure 3 Transgenic mice were screened using the QIAGEN Multiplex PCR Kit and sets of 3 primers to distinguish wild-type (wt), heterozygous mutant (ht), and homozygous mutant (hm) mice. A Using a primer set for the recombination activating gene 2 locus. B Using a primer set for the interferon- Ygene locus. M: markers. (Data kindly provided by S. zur Lage and S. Weiss, National Research Center for Biotechnology, Braunschweig, Germany.)




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