RNAi and OriGene siRNA
RNA interference is a gene-silencing phenomenon in which double-stranded RNAs cause the degradation of a homologous mRNA. Specific dsRNAs are reduced into small interfering RNA (siRNA), which serve as a guide for cleavage of homologous mRNA in the RNA- induced silencing complex (RISC). Degradation of a targeted mRNA causes specific suppression of that individual gene
OriGene has created a line of RNAi products, Trilencer-27 siRNA and HuSH-29 shRNA, to address the need to silence genes. Once transfected into a host cell, the siRNA duplex or shRNA cassette will work within the RNA interference complex to degrade its targeted mRNA and silence the gene
RNAi is regularly used to resolve questions regarding gene function, pathway elucidation, validation of gene and protein expression analysis, and pre- and post-transcriptional functions of gene related to disease. There are a number of ways to determine how an RNAi has affected the cell including measurement of mRNA levels, protein levels, and cellular functions. Selecting the appropriate assay for RNAi measure is important in generating meaningful knockdown results.
Tips for Successful Gene Knockdown
Measure of mRNA levels through qRT-PCR (quantitative reverse transcription - polymerase chain reaction) is very sensitive and rapid. QRT-PCR provides a pre-transcriptional measure of non-degraded mRNA available in a cell sample. The detectable mRNA levels for a gene silenced by siRNA should be lower than an untreated control sample
A common assay used to measure RNAi knockdown is by western blot assay and levels of protein expression. Using a protein specific antibody for the gene being silenced one can evaluate silencing, post-transcription. Cell lysates are run on a gel to separate proteins by size. The protein is then transferred to a membrane, which is probed with the antibody. Lower levels of detected protein as compared to an untreated cell extract control are indicative of successful gene silencing.
Cellular function assays can be as simple as measuring for a general effect like increased or decreased proliferation, apoptosis, or more complex phenotypic changes. There are a number of commercially available kits that can be used for measuring proliferation and apoptosis in cells. Phenotypic measures generally require more complex and specialized methods of evaluation, such as the function of a known pathway or other interaction.
A valid and successful knockdown experiment depends on a number of critical factors: Good design of the targeting sequence, high quality DNA, , the transfection method, inclusion of appropriate controls, and methods for the determination of knockdown. To properly control and measure your knockdown application, please consider the following points
Use negative controls
For siRNA experiment, it is important to demonstrate that the effect of a targeting duplex is gene specific, and not due to non-specific effects such as the interferon response or off-target silencing. Our negative control can be used to exclude these non-specific responses. All OriGene siRNA kits include a validated negative control duplex that is not present in the human, mouse, or rat genomes.
Use positive controls
The best control for a good transfection is demonstration of successful suppression of a control gene using a known effective siRNA. OriGene offers a validated HPRT1 Positive control duplex for this purpose. HPRT1 Positive control is a potent Dicer-Substrate duplex that has been shown to suppress HPRT mRNA levels >90% when used at 10 nM concentration.
Measure knockdown effectiveness
There are a number of methods used to measure gene knockdown effectiveness. Measurement of control genes as internal standards will help in evaluating and validating a successful and specific gene expression knockdown assay. Controls genes such as GAPDH, actin, or tubulin have been routinely used for this purpose. Measures of gene expression knockdown can be gathered using qRT-PCR and western blot analysis, for example. These methods are routinely used and have been described in publication many times.