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 ena
bles 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 multipl
ex 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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Custom and library siRNA for efficient gene silencing2.
Custom siRNA Oligo Synthesis Service3.
Ambion Custom4.
Ambion Custom: Made-to-Order5.
Custom RNA & RNA Isolation Services to Meet Your Specific Needs6.
Cancer siRNA Oligo Set Version 1.07.
Library siRNA8.
Efficient RNAi-mediated gene silencing in neuronal cells using QIAGEN
siRNA and TransMessenger Transfection Reagent*9.
Quantification of siRNA Silencing Efficiency
Using the LightCycler System10.
Housekeeping Genes: Universal Positive
Controls in siRNA Knockdown Experiments11.
Confirming gene silencing
mechanism by pGFP/GFP22
siRNA co-transfection