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RNAi Human/Mouse Starter Kit

For easy establishment of RNAi
  • A complete set of controls to establish and optimize RNAi
  • High transfection efficiency with low siRNA concentrations
  • Easy transfection optimization by observing cell death
  • Flexible tools available for knockdown validation

The RNAi Human/Mouse Starter Kit allows easy establishment of siRNA-mediated RNAi, optimization of transfection conditions, and routine control experiments. It includes AllStars Negative Control siRNA, which is the most thoroughly tested and validated negative control siRNA available, MAPK1 positive control siRNA, AllStars Hs Cell Death Control siRNA, and HiPerFect Transfection Reagent.

The RNAi Human/Mouse Starter Kit is intended for molecular biology applications. This product is not intended for the diagnosis, prevention, or treatment of a disease.
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Quantitative RT-PCR shows effective MAPK1 knockdown.
[A] HeLa cells, [B]  MCF-7 cells, and [C] NIH/3T3 cells were transfected with different concentrations of positive control MAPK1 siRNA and with nonsilencing siRNA. After 48 hours, cells were lysed and total RNA was isolated using the RNeasy Mini Kit. Quantitative, real-time RT-PCR was carried out using QuantiTect Kits. Expression of MAPK1 was normalized to the expression of GAPDH and quantified using a standard curve prepared using RNA from untreated cells.
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Expression of MAPK1 protein in several cell lines.

Western blot analysis shows the expression of MAPK1 in a variety of human and mouse cell lines. Tubulin was also detected using anti-tubulin primary antibody and was used as an internal loading control.

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Western blot analysis shows effective MAPK1 knockdown.

MCF-7 cells were transfected with positive control siRNA targeted against a sequence common to human and mouse MAPK1 mRNA or with nonsilencing siRNA (Control siRNA) using HiPerFect Transfection Reagent. After transfection and incubation, protein expression was determined by western blot analysis. Untransfected cells were also analyzed.

Performance
The RNAi Human/Mouse Starter Kit provides highly effective and tested reagent and siRNAs ensuring successful control experiments (see figures "Western blot analysis shows effective MAPK1 knockdown" and "Quantitative RT-PCR shows effective MAPK1 knockdown").
Principle

Control and optimization experiments are necessary for start-up RNAi experiments or for establishment of RNAi in a new cell line. In addition, data from appropriate control experiments are important for thorough, rigorous analysis of the results of RNAi experiments. A nonsilencing negative control is useful as a control for nonspecific silencing effects. A positive control, consisting of siRNA that is known to provide high levels of gene knockdown, can be routinely transfected in parallel with the siRNA under study to ensure that optimal conditions are maintained and to confirm experimental results. The RNAi Human/Mouse Starter Kit provides positive and negative control siRNAs for easy control and start-up experiments in both human and mouse cell lines. In addition, a cell-death phenotype control for human cells is included. This allows easy establishment and monitoring of transfection conditions.

Highly validated siRNA is used as a negative control and to monitor transfection

The RNAi Human/Mouse Starter Kit includes AllStars Negative Control siRNA. AllStars Negative Control siRNA is the most thoroughly tested and validated negative control siRNA currently available. This siRNA has no homology to any known mammalian gene. It has been validated using Affymetrix GeneChip arrays and a variety of cell-based assays and shown to exhibit minimal nonspecific effects on gene expression and phenotype. Cloning experiments confirmed that AllStars Negative Control siRNA enters RISC.

MAPK1 siRNA is a highly suitable positive control

A validated siRNA directed against a sequence common to both human and mouse MAPK1 mRNA is included in the kit. The protein kinase MAPK1 (also called Erk2 and MAPK2) is expressed in a wide range of human and mouse cell lines. Western blot analysis of human and mouse cell lysates showed that MAPK1 is ubiquitously expressed (see figure "Expression of MAPK1 protein in several cell lines"). The positive control siRNA provided in the kit is a highly pure siRNA that has 100% homology with both human and mouse MAPK1 gene sequences. The MAPK1 positive control siRNA almost eliminates MAPK1 expression in human and mouse cell lines (see figures "Western blot analysis shows effective MAPK1 knockdown" and "Quantitative RT-PCR shows effective MAPK1 knockdown"). It can be routinely transfected to ensure that experimental conditions remain consistent.

Procedure
Easy transfection monitoring by observation of cell death

AllStars Hs Cell Death Control siRNA is a blend of highly potent siRNAs targeting ubiquitously expressed human genes that are indispensable for cell survival. Knockdown of these genes induces a high degree of cell death that is visible by light microscopy. AllStars Hs Cell Death Control siRNA is a useful tool for siRNA transfection optimization and for routine use as a positive control in human cells.

Convenient measurement of gene silencing

Quantitative RT-PCR allows knockdown measurement at the mRNA level (see figure "Quantitative RT-PCR shows effective MAPK1 knockdown"). QIAGEN offers QuantiTect Primer Assays, which are ready-to-use, validated primer sets for highly sensitive and accurate quantification in SYBR® Green based real-time RT-PCR. QuantiTect Primer Assays and siRNAs for every human, mouse, and rat gene can be conveniently ordered online at GeneGlobe.

High transfection efficiency using low siRNA concentrations

The RNAi Human/Mouse Starter Kit includes HiPerFect Transfection Reagent, which has been developed for highly efficient siRNA transfection and knockdown in eukaryotic cells using low siRNA concentrations. Research suggests that off-target effects, which may produce misleading results in RNAi experiments, can be largely avoided by using low siRNA concentrations.

Applications
Establishment of RNAi in start-up experiments or for a new cell line
Transfection optimization
Routine control experiments
Features
Specifications
Applications Routine control experiments, transfection optimization...
Cell type Human and mouse cells
Controls Positive control for mouse and human MAPK1, nonsilencing control siRNA
Features High transfection efficiency with low siRNA concentrations
Nucleic acid siRNA
Number of possible transfections >160 transfections per kit
Transfection type Transient transfection
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