RNAi and OriGene HuSH
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 HuSH cassette expresses a gene specific short hairpin RNA that 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.
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 shRNA 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.
Co-transfection with reporter gene like luciferase or GFP can be used to optimize the transfection or infection efficiency of the shRNA construct. There are also commercially available validation vectors that provide an inverse signal to that of the shRNA is being expressed within the cell. The reporter is destroyed as the RNA produced by the shRNA expression cassette is being processed by the RNA interference mechanism.
Tips for Successful Gene Knockdown
A valid and successful knockdown experiment depends on a number of critical factors: Good design of the targeting sequence, high quality DNA, an efficient silencing vector, 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 antibiotic selection
One way to assure relevant data collection is to take advantage of the pRS puromycin selection marker to generate stable cell lines. Transient transfection does not guarantee a high percentage of cells expressing shRNA. This can simplify data analysis and eliminate considerable error from the experiment.
Use negative controls
For any shRNA experiment, it is important to demonstrate that the effect of a targeting construct is gene specific, and not due to non-specific effects such as the interferon response or off-target silencing. Our negative control plasmids can be used to exclude these non-specific responses.
All OriGene shRNA products include two negative controls. One is the original, empty pRS vector (TR20003) and the other is the non-effective shGFP control (TR30003) containing an shRNA cassette cloned into the same pRS vector. Use of an empty pRS vector demonstrates that a silencing effect is not due to interferon response. The non-effective shGFP plasmid expresses hairpin shRNA and serves as a negative control for gene specific knock down.
Please use both as negative controls in all your experiments to compare effects with the gene specific shRNA vectors. A valid test includes the use of both negative control plasmids. It would be unwise to include only one of the two negative controls or simply use the non-transfected parental cell as the negative control for the comparison, as cellular conditions may change dramatically upon transfection resulting is substantial assay bias.
Use positive controls
Positive control shRNA expression vectors against green fluorescent protein (GFP) and luciferase (Luc) genes are available for purchase (Catalog Numbers TR30001 and TR30002 respectively)
to use as knockdown controls for your experiments. Both shRNA-GFP and shRNA-Luc are constructed in the same plasmid vector, pRS, and have been shown to inhibit their respective
target genes by up to 90%. When co-transfected with GFP or luciferase expressing plasmids, these positive controls are readily assayed for establishment of the optimal transfection conditions for shRNA silencing in your cells.
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.
Protocols and Procedures
Plasmid DNA amplification with chemically competent cells or electrocompetent cells
Introduction of gene specific shRNA into mammalian cells via transient transfection
Creating a stable cell line expressing gene specific shRNA via transfection
Infection of gene specific shRNA into cells via retrovirus
Positive and Negative Controls - shRNA
Positive and Negative Controls - siRNA